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 ENISO10712:1995 has the status of a British Standard ICS 13.060.40BSEN ISO10712:1996 This British Standard, having
2、been prepared under the directionof the Health and Environment Sector Board, waspublished under the authorityof the StandardsBoardand comes into effect on 15April1996 BSI 03-2000 The following BSI references relate to the work on this standard: Committee reference EH/3/5 Draft for comment 93/505847
3、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 Subcommittee EH/3/5, Biological methods, upon which the following bodies were represented: BLWA Ltd. (The Association of the La
4、boratory Supply Industry) Department of Economic Development (Northern Ireland) Department of Health Department of the Environment (Water Directorate) Freshwater Biological Association Institution of Water and Environmental Management Ministry of Agriculture, Fisheries and Food National Rivers Autho
5、rity Royal Society of Chemistry Scottish Natural Heritage Soap and Detergent Industry Association Water Research Centre Water Services Association of England and Wales Amendments issued since publication Amd. No. Date CommentsBSEN ISO10712:1996 BSI 03-2000 i Contents Page Committees responsible Insi
6、de front cover National foreword ii Foreword 2 Introduction 3 1 Scope 3 2 Normative reference 3 3 Definitions 3 4 Principle 4 5 Reagents 4 6 Materials and equipment 5 7 Treatment of samples 5 8 Procedure 5 9 Validity criteria 7 10 Calculation of results 7 11 Expression of results 8 12 Test report 8
7、13 Interpretation of the results 8 14 Characteristics of the procedure 8 Annex A (informative) Procedure for testing substances soluble in water 9 Annex B (informative) Bibliography 9 Annex ZA (normative) Normative references to international publications with their relevant European publications 10
8、 Table 1 Final concentrations in the various media 5 Table 2 Example of a test series 7 Table 3 Example of test results 7 Table 4 Example of tabular presentation of results 8 List of references Inside back coverBSEN ISO10712:1996 ii BSI 03-2000 National foreword This British Standard has been prepar
9、ed by Technical Committee EH/3/5 and is the English language version of ENISO10712:1995 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 ISO10712:1995 pu
10、blished 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 immun
11、ity from legal obligations. Cross-references Publication referred to Corresponding British Standard BS 6068 Water quality ISO 7027:1990 Section 2.13:1994 Determination of turbidity Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, theEN ISO title page, pa
12、ges 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.EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 10712 December 1995 ICS 1
13、3.060 Descriptors: Water, quality, tests, water tests, biological tests, determination, toxicity, turbidimetric analysis English version Water quality Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test) (ISO 10712:1995) Qualit de leau Essai dinhibition de la c
14、roissance de Pseudomonas putida (essai dinhibition de la multiplication des cellules de Pseudomonas) (ISO 10712:1995) Wasserbeschaffenheit Pseudomonas putida Wachstumshemmtest (Pseudomonas-Zellvermehrungshemmtest) (ISO10712:1995) This European Standard was approved by CEN on1995-12-13. CEN members a
15、re 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-date lists and bibliographical references concerning such national standards may be obtained on application to
16、 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 responsibility of a CEN member into its own language and notified to the Central Secretariat has the same s
17、tatus 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 European Committee for Standardization
18、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 means reserved in all countries to CEN and its members. Ref. No. ENISO10712:1995 EENISO10712:1995 BSI 03-200
19、0 2 Foreword The text of the International Standard ISO10712:1995 has been prepared by Technical Committee ISO/TC147, Water quality, in collaboration with CEN/TC230, Water analysis. This European Standard shall be given the status of a national standard, either by publication of an identical text or
20、 by endorsement, at the latest by June1996, and conflicting national standards shall be withdrawn at the latest by June1996. In accordance with the CEN/CENELEC Internal Regulations, the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Ger
21、many, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, United Kingdom.ENISO10712:1995 BSI 03-2000 3 Introduction The bacterium Pseudomonas putida is used as an organism representative of heterotropic microorganisms in fresh water. 1 Scope This I
22、nternational 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 substances which react with the nutri
23、ent 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 (seeAnnex A). 2 Normative reference The
24、 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 on this International Standard are
25、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 Definitions For the purposes of this Interna
26、tional 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 relationship is graphically represe
27、nted 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 putida within16h 1h, compared to that
28、 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 by10% or50% respectively, compared to that of the control batch 3.4 stock culture bacterial culture obtained from the coll
29、ection 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 exponentially multiplying bacteria as an inoculum for t
30、he 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, dilution water and nutrient solution (witho
31、ut 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, nutrient solution and inoculum (without test sampl
32、e) 3.13 formazine nephelometric unit (FNU) formazine turbidity units. The optical density of a bacterial cell suspension at2=436nm, measured as formazine nephelometric units according to ISO7027ENISO10712:1995 4 BSI 03-2000 4 Principle Determination of the inhibitory effect of the sample on Pseudomo
33、nas putida by measurement of cell growth under the influence of varying dilutions of the test sample, compared to the cell growth of a culture obtained under the same conditions, but without the test sample. Determination of the cell concentration as optical density after a test period of16h 1h. The
34、 concentrations of the test sample at which cell multiplication is inhibited by10% and50% within16h 1h.are the basis for assessment. 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
35、 of the Pseudomonadaceae family; mobile rods (diameter0,74mto1,14m, length2,04m to4,04m) with polar flagellation. It occurs ubiquitously in soil and surface water. The optimal growth temperature is between25 C and30 C. NOTE 2The two following strains are suitable for this test: a) MIGULA, Berlin33/2
36、 strain (DSM50026) This strain is available from the following collection: German collection of microorganisms Mascheroder Weg 1b D-38124 Braunschweig Germany b) NCIB strain9494 This strain is available from the following collection: Torry Research Station P.O. Box 31 Aberdeen, UK Any other strain o
37、f equivalent sensitivity may be suitable. 5.2 Hydrochloric acid, c(HCl)=1mol/l. 5.3 Sodium hydroxide solution, c(NaOH)=1mol/l. More diluted or concentrated acids and alkaline solutions are permissible to adjust the pH as necessary. 5.4 Nutrient solution Prepare the stock solutionsI-IV (see5.4.1 to5.
38、4.4) and then sterilize them, for example at121 C for10min. The solutions can be stored for several weeks in the refrigerator at2 C to4 C. 5.4.1 Solution I Dissolve the following in water and dilute to500ml. 10,0g of sodium nitrate (NaNO 3 ) 2,40g of dipotassium hydrogen phosphate (K 2 HPO 4 ) 1,20g
39、 of potassium dihydrogen phosphate (KH 2 PO 4 ) 1,0 g of yeast extract 5.4.2 Solution II Dissolve the following in water and dilute to500ml. 10,0g of sodium nitrate (NaNO 3 ) 2,40g of dipotassium hydrogen phosphate (K 2 HPO 4 ) 1,20g of potassium dihydrogen phosphate (KH 2 PO 4 ) 5.4.3 Solution III,
40、 glucose solution Dissolve the following in water and dilute to500ml. 40,0 g of D(+)-glucose monohydrate (C 6 H 12 O 6 H 2 O) for biochemical and microbiological use 5.4.4 Solution IV, magnesium sulfate-iron(III) citrate solution Dissolve the following in water and dilute to1000ml. 4,0 g of magnesiu
41、m sulfate heptahydrate (MgSO 4 7H 2 O) 0,01 g of granulated iron(III) citrate NOTE 3In order to reduce the number of steps involved, solutionsI andIII can be combined, following sterilization, for the procedure in5.5 and8.1 and solutionsII andIII for the procedure in8.2. 5.5 Stock culture (seeTable
42、1) 5.5.1 Nutrient medium for the stock culture (slant agar) Dissolve18g of agar (high purity quality for microbiology) in water by heating. Add50ml of solutionI (5.4.1),125ml of solutionIII(5.4.3),100ml of solutionIV(5.4.4) and dilute to1000ml with water. Dispense6ml to10ml portions of the nutrient
43、medium into culture tubes while still liquid, close the tubes with plugs and sterilize for10min at121 C. Allow the nutrient medium to gel at a slant and store at2 C to4 C. 5.5.2 Handling of stock culture Store stock cultures of the test strain Pseudomonas putida in slant-agar culture tubes on the so
44、lid nutrient medium for stock cultures(5.5.1).ENISO10712:1995 BSI 03-2000 5 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 for24h at25 C 4 C (and store at25 C 4 C) for this purpo
45、se. 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 for24h. This is normal and does not indicate contamination. 6 Materials and equi
46、pment 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 befor
47、e 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 procedure, providing that this method is sufficiently sensitive and if it can be shown that the correlation with turbidi
48、metry is acceptable. 6.2 Microscope of minimum magnification 100 6.3 pH-meter 6.4 Temperature-controlled cabinet 6.5 Culture flasks 6.6 Autoclave 7 Treatment of samples Test the samples as soon as possible after collection and preparation. If unavoidable, preserve the samples by cooling (up to two d
49、ays at2 C to4 C) or freezing (up to two weeks at18 C). Preserve samples only in exceptional cases, because the toxicity of the sample can change on standing. Thoroughly shake and, if necessary, homogenize the sample before preparing the test medium. Measure the pH of the sample. In general, the test is carried out without adjusting the pH. If there are indications that an inhibitory effect is caused merely by an extreme pH, carry out an additional test in which the pH is adjusted to7,4. In this case
