ISO/TS 22939:2019
(Main)Soil quality — Measurement of enzyme activity patterns in soil samples using fluorogenic substrates in micro-well plates
Soil quality — Measurement of enzyme activity patterns in soil samples using fluorogenic substrates in micro-well plates
This document specifies a method for the measurement of several enzyme activities (arylsulfatase, α −glucosidase, β -glucosidase, Cellubisidase, β -Xylosidase, phosphodiesterase (PDE), chitinase, phosphomonoesterase (PME), leucine-aminopeptidase, Alanine-aminopeptidase) simultaneously (or not) using fluorigenic substrates 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 impacts 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.
Qualité du sol — Mesure en microplaques de l'activité enzymatique dans des échantillons de sol en utilisant des substrats fluorogènes
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Standards Content (Sample)
TECHNICAL ISO/TS
SPECIFICATION 22939
Second edition
2019-08
Soil quality — Measurement of
enzyme activity patterns in soil
samples using fluorogenic substrates
in micro-well plates
Qualité du sol — Mesure en microplaques de l'activité enzymatique
dans des échantillons de sol en utilisant des substrats fluorogènes
Reference number
ISO/TS 22939:2019(E)
©
ISO 2019
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ISO/TS 22939:2019(E)
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ISO/TS 22939:2019(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 1
5 Principle . 1
6 Reagents . 2
6.1 Buffers . 2
6.2 Substrates and standards . 3
6.2.1 Preparation of standard solutions . 3
6.2.2 Preparation of substrate solutions . 3
6.2.3 Preparation of multi-well plates . 3
6.2.4 Preparation of standard plates . 3
6.2.5 Preparation of substrate plates . 4
6.2.6 Fluorogenic substrates . 4
7 Apparatus and materials. 5
8 Procedure. 6
8.1 Sampling . 6
8.2 Sample preparation . 6
8.2.1 Homogenization . 6
8.2.2 Preparation of dilutions . 6
8.2.3 Sample distribution . 7
8.3 Incubation . 7
8.4 Fluorescence measurements . 7
9 Calculation of results . 7
10 Expression of results . 7
11 Test report . 8
Annex A (informative) Guidance on the use of freshly prepared substrates .9
Annex B (informative) Example of a graph for calculation .11
Bibliography .13
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ISO/TS 22939:2019(E)
Foreword
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 ISO technical committees. Each member body interested in a subject for which a technical
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ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
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.org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological characterization.
This second edition cancels and replaces the first edition (ISO/TS 22939:2010), which has been
technically revised. The main changes compared to the previous edition are as follows:
— Clause 3 “Terms and definitions” added;
— 6.2.4: unit corrected in (40 ml to 40 µl);
— 6.2.6, Table 1 (Chitinase change E.C. 3.2.1.30 to E.C.3.2.1.52 and Alanin-aminopeptidase E.C.
3.4.11.12 to E.C. 3.4.11.2).
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
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ISO/TS 22939:2019(E)
Introduction
Micro-organisms are responsible for many key processes in the cycle of elements. Enzymes play
key roles in the degradation and mineralization of organic macromolecules. The main postulate is
the microbial origin of soil enzymes, even if plant root exudates include enzymes. The simultaneous
monitoring of several enzyme activities important in the biodegradation of organic compounds
and mineralization of C, N, P and S in soil may reveal harmful effects caused by chemicals and other
anthropogenic impacts (e.g. acidification, compaction). However, the measurements carried out under
selected laboratory conditions using artificial substrates cannot be a substitute for the actual rate of
enzymatic processes in soil in situ.
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TECHNICAL SPECIFICATION ISO/TS 22939:2019(E)
Soil quality — Measurement of enzyme activity patterns
in soil samples using fluorogenic substrates in micro-
well plates
1 Scope
This document specifies a method for the measurement of several enzyme activities (arylsulfatase,
α −glucosidase, β -glucosidase, Cellubisidase, β -Xylosidase, phosphodiesterase (PDE), chitinase,
phosphomonoesterase (PME), leucine-aminopeptidase, Alanine-aminopeptidase) simultaneously (or
not) using fluorigenic substrates 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 impacts 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 documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 18400-206, Soil quality — Sampling — Part 206: Collection, handling and storage of soil under aerobic
conditions for the assessment of microbiological processes, biomass and diversity in the laboratory
ISO 10390, Soil quality — Determination of pH
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary
analysis)
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
4 Abbreviated terms
E.C. Enzyme code number defined by the Nomenclature Committee of the International Union of
Biochemistry and Molecular Biology (NC-IUBMB)
SOM Soil organic matter content
MUB Modified universal buffer
5 Principle
This document describes a method for the simultaneous measurements of several enzymes in soil
samples. It is based on the use of soil samples diluted in buffer containing fluorogenic substrates, which
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ISO/TS 22939:2019(E)
are incubated for 3 h at (30 ± 2) °C in multi-well plates. After the incubation the enzyme activities are
[1][2]
measured as fluorescence with a plate-reading fluorometer . The method described is based on
dried standard 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 the use of freshly prepared substrates is that an instrument for lyophilization is not
required.
6 Reagents
6.1 Buffers
6.1.1 General
The selection of the buffer depends on the soil sample because the pH strongly affects enzyme
activities. 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 of substrates
at different buffers needs to be ensured. Good stability has been observed in 0,5 mol/l sodium acetate
[3]
buffer at pH 5,5 .
6.1.2 Sodium acetate buffer, 0,5 mol/l, pH 5,5.
— sodium acetate trihydrate (CAS N°: 6131-90-4 – 136,08 g/mol): 68,04 g;
— deionized water 1 000 ml;
— acetic acid (CAS N°: 64-19-7 – 60,05 g/mol): >99,8 %.
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.
[4]
6.1.3 Modified universal buffer (MUB) .
6.1.3.1 Stock solution
— tris(hydroxymethyl)aminomethane (CAS N°: 77-86-01 – 121,14 g/mol): 12,1 g;
— maleic acid (CAS N°: 110-16-7 – 116,07 g/mol): 11,6 g;
— citric acid (CAS N°: 77-92-9 – 192,12 g/mol): 14,0 g;
— boric acid (CAS N°: 10043-35-3 -61,83 mol/l): 6,3 g;
— sodium hydroxide (CAS N°: 1310-73-2 – 40,00 g/mol): (1 mol/l) 488 ml;
— deionized water 1 000 ml.
Dissolve the ingredients and store the solution in a refrigerator.
6.1.3.2 Final buffer
— hydrochloric acid (CAS N°: 7647-01-0 -36,46 g/mol): (0,1 mol/l);
— sodium hydroxide (CAS N°: 1310-73-2 – 40,00 g/mol): (0,1 mol/l).
Place 200 ml of the stock solution (6.1.3.1) 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
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ISO/TS 22939:2019(E)
hydroxide. Adjust the volume to 1 000 ml with deionized water. Sterilize in an autoclave at (121 ± 3) °C
for 20 min.
6.2 Substrates and standards
6.2.1 Preparation of standard solutions
6.2.1.1 4-Methylumbelliferone (MUF) solution
— 4-methylumbelliferone (MUF) (CAS N°: 90-33-5 - 176,17 g/mol): 0,022 g;
— dimethylsulfoxide (DMSO) (CAS N°: 67-68-5 – 78,13 g/mol): add 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.
6.2.1.2 7-Amino-4-methylcoumarin (AMC) solution
— 7-amino-4-methylcoumarin (AMC) (CAS N°: 26093-31-2 – 175,18 g/mol): 0,021 9 g;
— dimethylsulfoxide (DMSO) (CAS N°: 67-68-5 – 78,13 g/mol): add 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.
6.2.2 Preparation of substrate solutions
Commercially 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 µmol/l, 2 500 µmol/l
or 2 750 µmol/l concentration in a volume of, for example, 50 ml, avoiding exposure to light. Weigh the
powder into a brown 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 µl) to
eight rows. To facilitate the use of these instruments enabling good volumetric precision, 2 500 µmol/l
solutions of the substrates should be prepared. However, for 4-MUF-β-d-glucopyranoside and for 4-MUF-
phosphate substrates, a solution with the centration of 2 750 µmol/l is needed in order to produce the
same final concentration of 500 µmol/l. These two solutions are further diluted simultaneously with
the addition of the sample; 20 µl dimethylsulfoxide is added to the wells of these two substrates to
facilitate dissolution. For chitinase activity measurement, a lower concentration is needed in order
to avoid substrate inhibition, and the preparation of a solution with a concentration of 1 000 µmol/l
4-MUF-N-acetyl-β-d-glucosaminide can be used to produce the final concentration of 200 µmol/l.
6.2.3 Preparation of multi-well plates
The substrate and standard solutions are added to multi-well plates 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, standards and
multiwell plates. A separate multi-well plate for substrates and standards has proved to be convenient.
6.2.4 Preparation of standard plates
Adequate replicate measurements, e.g. three to four replicates, are necessary due to the small sample
volume. Standardization requires several concentrations of MUF or AMC, in replicate. Exposure to
light shall be avoided during the dilution of standards. Calculate the required volume that depends on
the number of samples and multi-well plates prepared. One example for the preparation of standards
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ISO/TS 22939:2019(E)
covering a wide range of enzyme activities is given below, but modifications can be made depending on
the range of enzyme activities in the samples studied.
The stock solution of MUF with a concentration of 5 mmol/l is used to produce the dilutions containing
1 000 µmol/l, 500 µmol/l, 250 µmol/l, 125 µmol/l, 50 µmol/l, 25 µmol/l and 5 µmol/l MUF. Distribute
the volumes needed (e.g. 40 µl) into a multi-well plate for concentrations of 0 nmol/well, 0,2 nmol/well,
1,0 nmol/well, 2,0 nmol/well, 5,0 nmol/well, 10 nmol/well, 20 nmol/well and 40 nmol/well, in replicate.
This step is critical for the measurement uncertainty.
NOTE 1 This set of stock solutions enables the use of automatic dispensers, which yield a significantly better
precision than manual pipetting.
The stock solution of AMC with a concentration of 5 mmol/l is used to produce the dilutions containing
250 µmol/l, 125 µmol/l, 50 µmol/l, 25 µmol/l, 5 µmol/l, 2,5 µmol/l and 0,5 µmol/l AMC. Distribute the
volumes needed (e.g. 40 µl) into a multi-well plate for concentrations of 0 nmol/well, 0,2 nmol/well,
1,0 nmol/well, 2,0 nmol/well,
...
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