1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationDD ISO/TS 22939:2010Soil quality Measurementof enzyme activity patterns insoil samples using fluorogenicsubstrates in micro-well platesDD ISO/TS 22939:2010 DRAFT FOR DEVELOPMENTN
2、ational forewordThis Draft for Development is the UK implementation of ISO/TS22939:2010.This publication is not to be regarded as a British Standard.It is being issued in the Draft for Development series of publicationsand is of a provisional nature. It should be applied on thisprovisional basis, so
3、 that information and experience of its practicalapplication can be obtained.Comments arising from the use of this Draft for Developmentare requested so that UK experience can be reported to theinternational organization responsible for its conversion toan international standard. A review of this pu
4、blication willbe initiated not later than 3 years after its publication by theinternational organization so that a decision can be taken on itsstatus. Notification of the start of the review period will be made inan announcement in the appropriate issue of Update Standards.According to the replies r
5、eceived by the end of the review period,the responsible BSI Committee will decide whether to support theconversion into an international Standard, to extend the life of theTechnical Specification or to withdraw it. Comments should be sentto the Secretary of the responsible BSI Technical Committee at
6、 BritishStandards House, 389 Chiswick High Road, London W4 4AL.The UK participation in its preparation was entrusted to TechnicalCommittee EH/4, Soil quality.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include
7、all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2010ISBN 978 0 580 66288 1ICS 13.080.30Compliance with a British Standard cannot confer immunity fromlegal obligations.This Draft for Development was published under the authority ofthe Standards Policy
8、and Strategy Committee on 31 October 2010.Amendments issued since publicationDate Text affectedDD ISO/TS 22939:2010Reference numberISO/TS 22939:2010(E)ISO 2010TECHNICAL SPECIFICATION ISO/TS22939First edition2010-03-15Soil quality Measurement of enzyme activity patterns in soil samples using fluoroge
9、nic substrates in micro-well plates Qualit du sol Mesure en microplaques de lactivit enzymatique dans des chantillons de sol en utilisant des substrats fluorognes DD ISO/TS 22939:2010ISO/TS 22939:2010(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing
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14、 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2010 All rights reservedDD ISO/TS 22939:2010ISO/TS 22939:2010(E) ISO 2010 All rights reserved iiiContents Page Foreword iv Introduction.v 1 Scope1 2 Normative references1 3
15、 Abbreviated terms .1 4 Principle1 5 Reagents.2 6 Apparatus and materials.5 7 Procedure.6 8 Calculation of results 7 9 Expression of results8 10 Test report8 Annex A (informative) Guidance on the use of freshly prepared substrates .9 Annex B (informative) Example of a graph for calculation.11 Biblio
16、graphy13 DD ISO/TS 22939:2010ISO/TS 22939:2010(E) iv ISO 2010 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
17、 ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborate
18、s closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards
19、. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. In other circumstances, particularly when there is an urgent market req
20、uirement for such documents, a technical committee may decide to publish other types of document: an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in an ISO working group and is accepted for publication if it is approved by more than 50 % of the mem
21、bers of the parent committee casting a vote; an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting a vote. An ISO/PAS or ISO/TS is reviewed after th
22、ree years in order to decide whether it will be confirmed for a further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an International S
23、tandard or be withdrawn. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO/TS 22939 was prepared by Technical Committee ISO/TC 190, Soil quality, S
24、ubcommittee SC 4, Biological methods. DD ISO/TS 22939:2010ISO/TS 22939:2010(E) ISO 2010 All rights reserved vIntroduction Micro-organisms are responsible for many key processes in the cycle of elements. Enzymes are responsible for the degradation of organic molecules and their mineralization. The ma
25、in postulate is the microbial origin of soil enzymes, even if plant root exudates include enzymes. Extracellular enzymes in soil play key roles in the biodegradation of organic macromolecules. The simultaneous monitoring of several enzyme activities important in the biodegradation of organic compoun
26、ds and mineralization of C, N, P and S in soil may reveal harmful effects caused by chemicals and other anthropogenic impacts. However, the measurements carried out under selected laboratory conditions using artificial substrates cannot be a substitute for the actual rate of enzymatic processes in s
27、oil in situ. DD ISO/TS 22939:2010DD ISO/TS 22939:2010TECHNICAL SPECIFICATION ISO/TS 22939:2010(E) ISO 2010 All rights reserved 1Soil quality Measurement of enzyme activity patterns in soil samples using fluorogenic substrates in micro-well plates 1 Scope This Technical Specification specifies a meth
28、od for the measurement of several enzyme activities simultaneously in soil samples. Enzyme activities of soil vary seasonally and depend on the chemical, physical and biological characteristics of soil. Its application for the detection of harmful effects of toxic chemicals or other anthropogenic im
29、pacts depends on the simultaneous comparison of enzyme activities in a control soil similar to the test soil, or on exposure tests with chemicals or treatments. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, onl
30、y the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 10381-6, Soil quality Sampling Part 6: Guidance on the collection, handling and storage of soil under aerobic conditions for the assessment of microbiological pr
31、ocesses, biomass and diversity in the laboratory ISO 10390, Soil quality Determination of pH ISO 10694, Soil quality Determination of organic carbon and total carbon after dry combustion (elementary analysis) 3 Abbreviated terms E.C. Enzyme code number defined by the Nomenclature Committee of the In
32、ternational Union of Biochemistry and Molecular Biology (NC-IUBMB) SOM Soil organic matter content MUB Modified universal buffer 4 Principle This Technical Specification describes a method for the simultaneous measurements of several enzymes in soil samples. It is based on the use of soil samples di
33、luted in buffer containing fluorogenic substrates, which are incubated for 3 h at (30 2) C in multi-well plates. After the incubation the enzyme activities are measured as fluorescence with a plate-reading fluorometer (References 1 and 2 in the Bibliography). The method described is based on dried s
34、tandard and substrate plates enabling storage and limiting bias due to differences between reagent batches, and also enabling comparison between reagent batches. Annex A describes a method utilizing freshly prepared reagents, which has a clearly defined and exact incubation period. The advantage of
35、the use of freshly prepared substrates is that an instrument for lyophilization is not required. DD ISO/TS 22939:2010ISO/TS 22939:2010(E) 2 ISO 2010 All rights reserved5 Reagents 5.1 Buffers 5.1.1 General The selection of the buffer depends on the soil sample because the pH strongly affects enzyme a
36、ctivities. Sodium acetate buffer, 0,5 mol/l, at pH 5,5 has been used for acid soils with a high organic matter content. The use of the modified universal buffer (MUB) at the pH of the soil sample gives the flexibility necessary for coverage of a broad spectrum of different soils. Adequate stability
37、of substrates at different buffers needs to be ensured. Good stability has been observed in 0,5 mol/l sodium acetate buffer at pH 5,5 (Reference 3 in the Bibliography). 5.1.2 Sodium acetate buffer, 0,5 mol/l, pH 5,5 sodium acetate trihydrate 68,04 g; deionized water, ad 1 000 ml; acetic acid 99,8 %.
38、 Dissolve sodium acetate trihydrate in water (e.g. 800 ml) and adjust the pH to 5,5 with concentrated acetic acid ( 99,8 %; pro-analysis). Fill up to 1 000 ml. Sterilize in an autoclave at (121 3) C for 20 min. Store in a refrigerator for a maximum of two weeks. 5.1.3 Modified universal buffer (MUB)
39、 (Reference 4) 5.1.3.1 Stock solution tris(hydroxymethyl)aminomethane 12,1 g; maleic acid 11,6 g; citric acid 14,0 g; boric acid 6,3 g; sodium hydroxide (1 mol/l) 488 ml; deionized water, ad 1 000 ml. Dissolve the ingredients and store the solution in a refrigerator. 5.1.3.2 Final buffer hydrochlori
40、c acid (0,1 mol/l); sodium hydroxide (0,1 mol/l). Place 200 ml of the stock solution (5.1.3.1) in a 500 ml beaker containing a magnetic stirring bar, and place the beaker on a magnetic stirrer. Set the required pH with hydrochloric acid or with sodium hydroxide. Adjust the volume to 1 000 ml with de
41、ionized water. Sterilize in an autoclave at (121 3) C for 20 min. DD ISO/TS 22939:2010ISO/TS 22939:2010(E) ISO 2010 All rights reserved 35.2 Substrates and standards 5.2.1 Preparation of standard solutions 5.2.1.1 4-Methylumbelliferone (MUF) solution 4-methylumbelliferone (MUF) 0,022 0 g; dimethylsu
42、lfoxide (DMSO), ad 25 ml. MUF in powder form can be stored at room temperature but protected from light. Weigh MUF carefully and dissolve it in DMSO in a brown volumetric flask, avoiding exposure to daylight. The solution cannot be stored. 5.2.1.2 7-Amino-4-methylcoumarin (AMC) solution 7-amino-4-me
43、thylcoumarin (AMC) 0,021 9 g; dimethylsulfoxide (DMSO), ad 25 ml. AMC as powder can be stored in the refrigerator. Weigh AMC carefully and dissolve it in DMSO in a brown volumetric flask, avoiding exposure to daylight. The solution cannot be stored. 5.2.2 Preparation of substrate solutions Commercia
44、lly available fluorogenic substrates are delivered as powders that can be stored deep-frozen at (20 2) C. On the day of use, weigh the amount required for a 1 000 mol/l, 2 500 mol/l or 2 750 mol/l concentration in a volume of, for example, 50 ml, avoiding exposure to light. Weigh the powder into a b
45、rown volumetric flask and fill to the required volume with DMSO. The volume should be big enough for reliable weighing and measurement of volumes. It also depends on the number of plates needed. The commonly used dispensers are able to distribute simultaneously just one volume (e.g. 40 l) to eight r
46、ows. To facilitate the use of these instruments enabling good volumetric precision, 2 500 mol/l solutions of the substrates should be prepared. However, for 4-MUF-D-glucopyranoside and for 4-MUF-phosphate substrates, a solution with the concentration of 2 750 mol/l is needed in order to produce the
47、same final concentration of 500 mol/l. These two solutions are further diluted simultaneously with the addition of the sample; 20 l dimethylsulfoxide is added to the wells of these two substrates to facilitate dissolution. For chitinase activity measurement, a lower concentration is needed in order
48、to avoid substrate inhibition, and the preparation of a solution with a concentration of 1 000 mol/l 4-MUF-N-acetyl-D-glucosaminide can be used to produce the final concentration of 200 mol/l. 5.2.3 Preparation of multi-well plates The substrate and standard solutions are added to multi-well plates
49、as solutions and dried (e.g. freeze-dried) on the multi-well plates directly after dispensing. Dry plates can be stored at (20 2) C for a year. Exposure to light shall be avoided during handling and storage of substrates and standards. A separate multi-well plate for substrates and standards has proved to be convenient. 5.2.4 Preparation of standard plates Adequate replicate measurements, e.g. three to four replicates, are necessary due to the small sample volume
