《BS-EN-ISO-10712-1996 BS-6068-5.19-1996.pdf》由会员分享，可在线阅读，更多相关《BS-EN-ISO-10712-1996 BS-6068-5.19-1996.pdf（16页珍藏版）》请在三一文库上搜索。
1、BRITISH STANDARD BS EN ISO 10712:1996 BS 6068-5.19: 1996 Water quality Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test) The European Standard EN ISO 10712:1995 has the status of a British Standard ICS 13.060.40 Licensed Copy: sheffieldun sheffieldun, na, Sa
2、t Nov 18 01:40:53 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS EN ISO 10712:1996 This British Standard, having been prepared under the direction of the Health and Environment Sector Board, was published under the authority of the Standards Board and comes into effect on 15 April 1996 BSI 03-2000 Th
3、e following BSI references relate to the work on this standard: Committee reference EH/3/5 Draft for comment 93/505847 DC ISBN 0 580 25598 0 Committees responsible for this British Standard The preparation of this British Standard was entrusted by Technical Committee EH/3, Water quality, to Subcommi
4、ttee EH/3/5, Biological methods, upon which the following bodies were represented: BLWA Ltd. (The Association of the Laboratory Supply Industry) Department of Economic Development (Northern Ireland) Department of Health Department of the Environment (Water Directorate) Freshwater Biological Associat
5、ion Institution of Water and Environmental Management Ministry of Agriculture, Fisheries and Food National Rivers Authority Royal Society of Chemistry Scottish Natural Heritage Soap and Detergent Industry Association Water Research Centre Water Services Association of England and Wales Amendments is
6、sued since publication Amd. No.DateComments Licensed Copy: sheffieldun sheffieldun, na, Sat Nov 18 01:40:53 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS EN ISO 10712:1996 BSI 03-2000i Contents Page Committees responsibleInside front cover National forewordii Foreword2 Introduction3 1Scope3 2Normati
7、ve reference3 3Definitions3 4Principle4 5Reagents4 6Materials and equipment5 7Treatment of samples5 8Procedure5 9Validity criteria7 10Calculation of results7 11Expression of results8 12Test report8 13Interpretation of the results8 14Characteristics of the procedure8 Annex A (informative) Procedure f
8、or testing substances soluble in water9 Annex B (informative) Bibliography9 Annex ZA (normative) Normative references to international publications with their relevant European publications10 Table 1 Final concentrations in the various media5 Table 2 Example of a test series7 Table 3 Example of test
9、 results7 Table 4 Example of tabular presentation of results8 List of referencesInside back cover Licensed Copy: sheffieldun sheffieldun, na, Sat Nov 18 01:40:53 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS EN ISO 10712:1996 ii BSI 03-2000 National foreword This British Standard has been prepared b
10、y Technical Committee EH/3/5 and is the English language version of EN ISO 10712:1995 Water quality Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test) published by the European Committee for Standardization (CEN). It was derived by CEN from ISO 10712:1995 pub
11、lished by the International Organization for Standardization (ISO). A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immuni
12、ty from legal obligations. Cross-references Publication referred toCorresponding British Standard BS 6068 Water quality ISO 7027:1990Section 2.13:1994 Determination of turbidity Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, the EN ISO title page, page
13、s 2 to 10, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover. Licensed Copy: sheffieldun sheffieldun, na, Sat Nov 18 01:40:53 GMT+00:00 2006, Unc
14、ontrolled Copy, (c) BSI EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 10712 December 1995 ICS 13.060 Descriptors: Water, quality, tests, water tests, biological tests, determination, toxicity, turbidimetric analysis English version Water quality Pseudomonas putida growth inhibition test (
15、Pseudomonas cell multiplication inhibition test) (ISO 10712:1995) Qualit de leau Essai dinhibition de la croissance de Pseudomonas putida (essai dinhibition de la multiplication des cellules de Pseudomonas) (ISO 10712:1995) Wasserbeschaffenheit Pseudomonas putida Wachstumshemmtest (Pseudomonas-Zellv
16、ermehrungshemmtest) (ISO 10712:1995) This European Standard was approved by CEN on 1995-12-13. 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 standard without any alteration. Up-to-da
17、te 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 version in any other language made by translation under the
18、 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, Ireland, Italy, Luxembourg, Netherlands,
19、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, B-1050 Brussels 1995 All rights of reproduction and communication in any form and by any
20、means reserved in all countries to CEN and its members. Ref. No. EN ISO 10712:1995 E Licensed Copy: sheffieldun sheffieldun, na, Sat Nov 18 01:40:53 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN ISO 10712:1995 BSI 03-2000 2 Foreword The text of the International Standard ISO 10712:1995 has been prep
21、ared by Technical Committee ISO/TC 147, Water quality, in collaboration with CEN/TC 230, Water analysis. 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 June 1996, and conflicting national standar
22、ds shall be withdrawn at the latest by June 1996. 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, Netherlands, Norway, Por
23、tugal, Spain, Sweden, Switzerland, United Kingdom. Licensed Copy: sheffieldun sheffieldun, na, Sat Nov 18 01:40:53 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN ISO 10712:1995 BSI 03-20003 Introduction The bacterium Pseudomonas putida is used as an organism representative of heterotropic microorgani
24、sms in fresh water. 1 Scope This International Standard specifies a test method for determining the inhibitory effect of surface, ground and waste water on Pseudomonas putida. This method is not suitable for highly coloured test samples, or samples containing undissolved or volatile materials or sub
25、stances which react with the nutrient solution, or which undergo changes during the test (for example by precipitation, or biochemical or photochemical degradation) and may give false results and/or impair the reproducibility. The method is also suitable for testing substances soluble in water (see
26、Annex A). 2 Normative reference The following standard contains provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publication, the edition indicated was valid. All standards are subject to revision, and parties to agreements based
27、on this International Standard are encouraged to investigate the possibility of applying the most recent edition of the standard indicated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 7027:1990, Water quality Determination of turbidity. 3 Definitio
28、ns For the purposes of this International Standard, the following definitions apply. 3.1 multiplication; growth increase in the number of cells during the test period 3.2 concentration-effect relationship dependence of cell multiplication inhibition on the concentration of the test sample NOTE 1The
29、relationship is graphically represented by plotting the inhibition values along the ordinate against the sample concentrations along the abscissa. 3.3 effective concentration (EC) concentration of the test sample giving a calculated or interpolated inhibition of cell multiplication of Pseudomonas pu
30、tida within 16 h 1 h, compared to that of the control batch the concentrations of test samples (EC10 and EC50) are determined from the concentration-effect relationship (3.2) at which cell multiplication is inhibited by 10 % or 50 % respectively, compared to that of the control batch 3.4 stock cultu
31、re bacterial culture obtained from the collection strain of the laboratory and intended to provide an inoculum for the preculture in the test procedure 3.5 preculture bacterial culture separately used to adapt the test bacteria to the test conditions and to produce an adequate number of exponentiall
32、y multiplying bacteria as an inoculum for the test culture 3.6 test culture inoculated test medium (3.9) 3.7 inoculum suspension of bacteria used to inoculate a nutrient solution 3.8 nutrient solution aqueous solution of nutrients required for bacterial growth 3.9 test medium mixture of test sample,
33、 dilution water and nutrient solution (without inoculum) 3.10 sample the surface, ground, or waste water to be tested 3.11 test sample the sample, after inclusion of all preparatory steps such as homogenization, pH adjustment, filtration, centrifugation 3.12 control mixture of dilution water, nutrie
34、nt solution and inoculum (without test sample) 3.13 formazine nephelometric unit (FNU) formazine turbidity units. The optical density of a bacterial cell suspension at 2= 436 nm, measured as formazine nephelometric units according to ISO 7027 Licensed Copy: sheffieldun sheffieldun, na, Sat Nov 18 01
35、:40:53 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN ISO 10712:1995 4 BSI 03-2000 4 Principle Determination of the inhibitory effect of the sample on Pseudomonas putida by measurement of cell growth under the influence of varying dilutions of the test sample, compared to the cell growth of a culture
36、 obtained under the same conditions, but without the test sample. Determination of the cell concentration as optical density after a test period of 16 h 1 h. The concentrations of the test sample at which cell multiplication is inhibited by 10 % and 50 % within 16 h 1 h. are the basis for assessment
37、. 5 Reagents Use chemicals of analytical grade and deionized water or water of equivalent purity. 5.1 Test organism Pseudomonas putida, a gram-negative aerobic bacterium of the Pseudomonadaceae family; mobile rods (diameter 0,7 4m to 1,1 4m, length 2,0 4m to 4,0 4m) with polar flagellation. It occur
38、s ubiquitously in soil and surface water. The optimal growth temperature is between 25 C and 30 C. NOTE 2The two following strains are suitable for this test: a) MIGULA, Berlin 33/2 strain (DSM 50026) This strain is available from the following collection: German collection of microorganisms Mascher
39、oder Weg 1b D-38124 Braunschweig Germany b) NCIB strain 9494 This strain is available from the following collection: Torry Research Station P.O. Box 31 Aberdeen, UK Any other strain of equivalent sensitivity may be suitable. 5.2 Hydrochloric acid, c(HCl) = 1 mol/l. 5.3 Sodium hydroxide solution, c(N
40、aOH) = 1 mol/l. More diluted or concentrated acids and alkaline solutions are permissible to adjust the pH as necessary. 5.4 Nutrient solution Prepare the stock solutions I-IV (see 5.4.1 to 5.4.4) and then sterilize them, for example at 121 C for 10 min. The solutions can be stored for several weeks
41、 in the refrigerator at 2 C to 4 C. 5.4.1 Solution I Dissolve the following in water and dilute to 500 ml. 10,0 g of sodium nitrate (NaNO3) 2,40 g of dipotassium hydrogen phosphate (K2HPO4) 1,20 g of potassium dihydrogen phosphate (KH2PO4) 1,0 g of yeast extract 5.4.2 Solution II Dissolve the follow
42、ing in water and dilute to 500 ml. 10,0 g of sodium nitrate (NaNO3) 2,40 g of dipotassium hydrogen phosphate (K2HPO4) 1,20 g of potassium dihydrogen phosphate (KH2PO4) 5.4.3 Solution III, glucose solution Dissolve the following in water and dilute to 500 ml. 40,0 g of D(+)-glucose monohydrate (C6H12
43、O6H2O) for biochemical and microbiological use 5.4.4 Solution IV, magnesium sulfate-iron(III) citrate solution Dissolve the following in water and dilute to 1 000 ml. 4,0 g of magnesium sulfate heptahydrate (MgSO47H2O) 0,01 g of granulated iron(III) citrate NOTE 3In order to reduce the number of ste
44、ps involved, solutions I and III can be combined, following sterilization, for the procedure in 5.5 and 8.1 and solutions II and III for the procedure in 8.2. 5.5 Stock culture (see Table 1) 5.5.1 Nutrient medium for the stock culture (slant agar) Dissolve 18 g of agar (high purity quality for micro
45、biology) in water by heating. Add 50 ml of solution I (5.4.1), 125 ml of solution III (5.4.3), 100 ml of solution IV (5.4.4) and dilute to 1 000 ml with water. Dispense 6 ml to 10 ml portions of the nutrient medium into culture tubes while still liquid, close the tubes with plugs and sterilize for 1
46、0 min at 121 C. Allow the nutrient medium to gel at a slant and store at 2 C to 4 C. 5.5.2 Handling of stock culture Store stock cultures of the test strain Pseudomonas putida in slant-agar culture tubes on the solid nutrient medium for stock cultures (5.5.1). Licensed Copy: sheffieldun sheffieldun,
47、 na, Sat Nov 18 01:40:53 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN ISO 10712:1995 BSI 03-20005 Table 1 Final concentrations in the various media Start new stock cultures at intervals of one week, to preserve the test strain. Incubate the inoculated stock cultures for 24 h at 25 C 4 C (and store
48、at 25 C 4 C) for this purpose. Long-term recultivation of one strain may cause changes in the sensitivity of the test organisms. In this case, a new culture of the test strain has to be used. NOTE 4A green pigment may be produced after incubation for 24 h. This is normal and does not indicate contam
49、ination. 6 Materials and equipment Equipment which comes into contact with the test material during preparation of the nutrient medium, or during the test period, shall consist of glass or another chemically inert material. All glass equipment and stoppers which come into contact with the test cultures shall be sterilized before use, if they are not sterilized together with the nutrient solutions. 6.1 Spectrometer or turbidimeter Alternatively, determine the state of growth of the culture by a different