1、EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 26461-1 January 1993 UDC 628.U.3 : 620.1 : 543.39 : 579.852.13 Descriptors: Water, quality, water tests, microbiological analysis, micro-organisms, sulfite reducing bacteria, clostridium English version Water quality - Detection and enumeration of
2、 the spores of sulfite-reducing anaerobes (clostridia) - Part 1 : Method by enrichment in a liquid medium (IS0 6461-1 1986) Qualit de leau - Recherche et dnombrement des spores de micro-organismes anarobies Anaerobier (Clostridien) - sulfito-rducteurs (clostridia) - Partie 1 : Mthode par enrichissem
3、ent dans un milieu liquide Wasserbeschaffenheit - Nachweis und Zhlung der Sporen sulfitreduzierender %il 1 : Flssigkeitsanreichening (IS0 6461-1 : 1986) (IS0 6461-1 : 1986) This European Standard was approved by CEN on 1993-01-20. CEN members are bound to comply with the CENKENELEC Internal Regulati
4、ons 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 Secretariat or to any CEN member. This Europ
5、ean 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 official versions. CEN members are the natio
6、nal 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 Europen de Normalisation Europisches Komitee fr Nor
7、mung Central Secretariat: rue de Stassart 36, B- 1050 Brussels O 1993 Copyright reserved to CEN members Ref. No. EN 26461-1 : 1993 E m CEN EN*26463-3 93 = 3404589 0042973 T44 Page 2 EN 26461-1 : 1993 Foreword This European Standard is the endorsement of IS0 6461-1. Endorsement of IS0 6461-1 was reco
8、mmended by Bchnical Committee 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 conflicti
9、ng national standards shall be withdrawn at the latest by July 1993. The standard was approved and 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, Ire
10、land, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, United Kingdom. CEN EN+2b4bL-L 93 = 3404589 0042972 980 Page 3 EN 26461-1 : 1993 Water quality - Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) - Part 1 : Method by enrichment in a
11、 liquid medium O Introduction The spores 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 Escherichia coli and other coliform organisms, the spores sur- vive in water for long period
12、s 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 resistant to chlorination at levels which are normally used for the treatment of water, and they are thus useful f
13、or 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 This part of IS0 6461 specifies a method for the detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) by enric
14、hment in a liquid medium. 2 Field of application The method 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. Part3: Guidance on the preservation
15、 and handling of samples. IS0 8199, Water quality - General guidance for microbiological examination by enumeration of micro- organisms on culture media.” 4 Definition For the purpose of this part of IS0 6461, the following defi- nition applies. clostridia : Sulfite-reducing, spore-forming, anaerobi
16、c 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 of a water sample requires the following steps. 5.1 Selection of spores Selection of spores in the sample by apply
17、ing heat for a period of time sufficient to destroy vegetative bacteria. 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 I 1 OC for 44 I 4 h in anaerobic condition
18、s. 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 basic components or complete dehydrated media be used. Similarly, commercially prepared reagents may a
19、lso be used. The manufacturers instructions shall be rigorously followed. The chemical products used for the preparation of the culture media and the reagents shall be of recognized analytical quality. 7 1) At present at the stage of draft. CEN EN*2b4b1-1 93 3404589 0042973 817 = Page 4 EN 26461-1 :
20、 1993 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 36961. Measurements of pH shall be made using a pH meter, measurements being referred to a temperature of 25 OC. if the prepared cultur
21、e 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 Culture media and diluent 6.2.1 Diluent Use one of the diluents given in IS0 8199. 6.2.2 Differential reinforced clostridial medium (DRCM1 6.2.2.1 Sin
22、gle strength basal medium Composition Peptone tryptic digest of meat Meat extract Yeast extract Starch Hydrated sodium acetate Glucose L-Cysteine hydrochloride Water Preperation Mix the peptone, meat extract, sodium acetate and yeast ex- tract with 800 ml of water. With the remaining 200 ml of disti
23、lled water, prepare a starch solution as follows: mix the starch in a little cold water to form a paste. Heat the 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.
24、 Finally, add the glucose and L-cysteine hydrochloride. Dissolve. Adjust the pH to 7,l to 7.2 with 1 mol/l sodium hydroxide. Transfer 25 ml aliquots of the medium into screw-capped bot- tles of capacity 25 ml. Sterilize in the autoclave at 121 I 1 OC for 15 min. 6.2.2.2 Double styength basal medium
25、Prepare the double strength medium as in 6.2.2.1 but reduce the volume of water by half. Transfer 10 ml and 50 ml aliquots of the medium into screw- capped bottles of capacities 25 ml and 100 ml respectively. 6.2.3 Sodium sulfite (Na2SO3). 4 % (m/m) solution. Dissolve 4 g of anhydrous sodium sulfite
26、 in 100 ml of water. Sterilize by filtration. Store at between 2 and 5 OC. It is advisable to prepare a fresh solution every 14 days. 6.2.4 Iron(llll citrate (C6H,0,Fe), 7 % (m/m) solution, Dissolve 7 g of iron(ll1) citrate in 100 ml of water. Sterilize by filtration. Store at between 2 and 5 OC. It
27、 is advisable to prepare a fresh solution every 14 days. 6.2.5 Complete medium 6.2.5.1 tions of sodium sulfite (6.2.3) and iron(lll) citrate (6.2.4). On the day of analysis, mix equal volumes of the solu- 6.2.5.2 Add 0,5 ml of the mixture (6.2.5.1) to each bottle of single strength medium (6.2.2.11,
28、 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 ml, and 2 ml to each 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
29、 25 ml. Screw-cap bottles or vials and stoppers of boron 7.2 Volumetric pipettes, of capacities 10 and 1 ml. 7.3 Water baths, thermostaticallv 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 81
30、99 for sampling techniques. 9 Procedure 9.1 Treatment of samples Refer to IS0 5667/3 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 f 5 OC for 15 min from the ti
31、me it reaches that temperature. A similar bottle containing the same volume CEN EN12b4bL-1 93 m 3404589 0042974 753 W Page 5 EN 26461-1 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 b
32、ottle 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 of the double strength complete medium (6.2.5.3). Add 10 ml of sample (9.2) to a series of five 25 ml screw-cap bottles containing 10 ml of double
33、 strength complete medium (6.2.5.3). Add 1 mi of 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). 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 sin
34、gle 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 MPN count, use a Mo 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, to
35、p 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 very small volume of air re- mains, then seal the bottles hermetically, or incubate under anaerobic conditions. Incubate the inoculated bottles at 37
36、f 1 OC for 44 k 4 h. Large volumes of culture in hermetically sealed glass bottles may explode due to gas production. The addition of iron wire, heated to redness and placed into the medium before inocula- tion, may aid anaerobiosis. 9.4 Interpretation Bottles in which blackening is observed, as a r
37、esult of the reduction of sulfite and the precipitation of iron(ll) 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 sul
38、fite-reducing anaerobes (clostridia) per volume of sample. It shall also men- tion any operating details not specified in this part of IS0 6461, or regarded as optional, together with details of any incidents likelv to have influenced the results. The test report shall include all the information necessary for the complete identification of the sample.
