EN ISO 20743:2007

SLOVENSKI STANDARD
01-julij-2007
Tekstilije - Ugotavljanje protibakterijske aktivnosti na izdelkih s protibakterijsko
apreturo (ISO 20743:2007)
Textiles - Determination of antibacterial activity of antibacterial finished products (ISO
20743:2007)
Textilien - Bestimmung der antibakteriellen Wirkung antibakteriell behandelter
Erzeugnisse (ISO 20743:2007)
Textiles - Détermination de l'activité antibactérienne des produits finis antibactériens
(ISO 20743:2007)
Ta slovenski standard je istoveten z: EN ISO 20743:2007
ICS:
59.080.01 Tekstilije na splošno Textiles in general
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 20743
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2007
ICS 59.080.01; 07.100.99
English Version
Textiles - Determination of antibacterial activity of antibacterial
finished products (ISO 20743:2007)
Textiles - Détermination de l'activité antibactérienne des Textilien - Bestimmung der antibakteriellen Wirkung
produits finis antibactériens (ISO 20743:2007) antibakteriell behandelter Erzeugnisse (ISO 20743:2007)
This European Standard was approved by CEN on 16 May 2007.
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-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN Management Centre 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 CEN Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2007 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20743:2007: E
worldwide for CEN national Members.

Foreword
This document (prEN ISO 20743:2007) has been prepared by Technical Committee ISO/TC 38
"Textiles" in collaboration with Technical Committee CEN/TC 248 "Textiles and textile products",
the secretariat of which is held by BSI.

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 December 2007, and conflicting national
standards shall be withdrawn at the latest by December 2007.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United
Kingdom.
Endorsement notice
The text of ISO 20743:2007 has been approved by CEN as EN ISO 20743:2007 without any
modifications.
INTERNATIONAL ISO
STANDARD 20743
First edition
2007-06-01
Textiles — Determination of antibacterial
activity of antibacterial finished products
Textiles — Détermination de l'activité antibactérienne des produits finis
antibactériens
Reference number
ISO 20743:2007(E)
©
ISO 2007
ISO 20743:2007(E)
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ii © ISO 2007 – All rights reserved

ISO 20743:2007(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Safety precaution. 2
5 Apparatus . 2
6 Reagents and culture media. 4
7 Reference strains. 8
7.1 Strains. 8
7.2 Storage of strains . 8
8 Quantitative measurement. 10
8.1 Plate count method. 10
8.2 Luminescence method. 11
9 Shaking method. 12
9.1 Shaking by vortex mixer . 12
9.2 Shaking by hand . 12
9.3 Shaking by Stomacher . 12
10 Test procedures . 12
10.1 Absorption method. 12
10.2 Transfer method. 17
10.3 Printing method . 21
Bibliography . 27

ISO 20743:2007(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 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 collaborates 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. 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.
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 20743 was prepared by Technical Committee ISO/TC 38, Textiles.
iv © ISO 2007 – All rights reserved

ISO 20743:2007(E)
Introduction
These test methods were established in order to address the substantial need for an International Standard to
determine antibacterial activity for antibacterial finished textile products.

INTERNATIONAL STANDARD ISO 20743:2007(E)

Textiles — Determination of antibacterial activity of
antibacterial finished products
1 Scope
This International Standard specifies quantitative test methods to determine the antibacterial activity of
antibacterial finished textile products including nonwovens.
This International Standard is applicable to all textile products, including cloth, wadding, thread and material
for clothing, home furnishings and miscellaneous goods regardless of the type of antibacterial agent used
(organic, inorganic, natural or man-made) or the method of application (built-in, after-treatment or grafting).
Based on the intended application and on the environment in which the textile product is to be used, the user
can select the most suitable of the following three methods on determination of antibacterial activity:
a) absorption method (an evaluation method in which test bacterial suspension is inoculated directly onto
samples);
b) transfer method (an evaluation method in which test bacteria are placed on an agar plate and transferred
onto samples);
c) printing method (an evaluation method in which test bacteria are placed on a filter and printed onto
samples).
The colony plate count method and the ATP (ATP = Adenosine Tri-phosphate) luminescence method are
also specified for measuring the enumeration of bacteria.
2 Normative references
The following referenced documents are indispensable for the application 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 6330, Textiles — Domestic washing and drying procedures for textile testing
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
textile
fabric
general term employed for designating textile surfaces, woven fabrics, knitted fabrics, etc., formed by the
interlocking of textile materials having a certain cohesion and which are generally intended for clothing or
furniture applications
NOTE Often includes certain types of nonwovens.
ISO 20743:2007(E)
3.2
control fabric
fabric used to validate the growth condition of test bacteria
NOTE The same fabric as the fabric to be tested but without antibacterial treatment. If this is not possible, then
100 % cotton fabric without fluorescent brighteners or other finish.
3.3
antibacterial agent
product designed to prevent or mitigate the growth of bacteria, to reduce the number of bacteria or to kill
bacteria
3.4
antibacterial finish
treatment designed to prevent or mitigate the growth of bacteria, to reduce the number of bacteria or to kill
bacteria
3.5
antibacterial activity
activity of an antibacterial finish used to prevent or mitigate the growth of bacteria, to reduce the number of
bacteria or to kill bacteria
3.6
plate count method
method in which the number of bacteria present after incubation is calculated by counting the number of
colonies according to a ten-time dilution method
NOTE The results are expressed in “CFU (Colony Forming Unit)”.
3.7
luminescence method
method in which the amount of ATP contained in bacterial cells is measured
NOTE The results are expressed in “mol of ATP”.
3.8
neutralizer
chemical agents used to inactivate, neutralize, or quench the antibacterial properties of antibacterial agents
4 Safety precaution
Test methods specified herein require the use of bacteria.
These tests should be carried out by persons with training and experience in the use of microbiological
techniques.
Appropriate safety precautions should be observed with due consideration given to country-specific
regulations.
5 Apparatus
Usual laboratory apparatus and, in particular, the following.
5.1 Spectrophotometer, capable of measuring at a 620 nm to 660 nm wavelength, or McFarland’s
nephelometer.
5.2 Incubator, capable of maintaining a constant temperature of 37 °C ± 2 °C.
2 © ISO 2007 – All rights reserved

ISO 20743:2007(E)
5.3 Water baths, one capable of maintaining a constant temperature of 46 °C ± 2 °C and another capable
of maintaining a temperature of 70 °C to 90 °C.
5.4 Mixer, producing a vortex shaking action.
5.5 Stomacher, capable of speeds of 6 blows/s to 8 blows/s, with the corresponding disposable containers.
5.6 Clean bench, for microbial test.
5.7 Washing machine, in accordance with the specifications of ISO 6330.
5.8 Humidity chamber, tropical chamber or other container capable of maintaining a high-humidity
atmospheric condition.
−13 −7
5.9 Luminescence photometer, capable of measuring ATP of 10 mol/l to 10 mol/l at 300 nm to
650 nm with a luminescence-measuring reagent.
5.10 Printing apparatus, capable of applying a 4 N load to a test piece and rotating the piece 180° in one
direction for a period of 3,0 s.
5.11 Refrigerator, capable of maintaining a temperature of between 2 °C and 8 °C.
5.12 Freezers, one adjustable to a temperature below −70 °C and another to a temperature below −20 °C.
5.13 Balance, which can be read to the nearest 0,01 g.
5.14 Filtering apparatus, consisting of an upper container equipped with a membrane filter and a lower
container equipped with a suction opening.
5.15 Pipette, having the most suitable volume for each use, with a tip made of glass or plastic, and with a
tolerance of 0,5 % or less.
5.16 Vials, 30 ml glass bottles, with screw openings, polytetrafluoroethylene or silicone packing and caps
made of polypropylene, polycarbonate, or other suitable material.
5.17 Petri dishes, that have been sterilized, made of glass or plastic, in diameter sizes of 90 mm to 100 mm
and 55 mm to 60 mm.
5.18 Glass rod, with a diameter of approximately 18 mm.
5.19 Anti-bumping granules (glass beads), with a diameter of 3 mm to 4 mm.
5.20 Erlenmeyer flask, capacity 100 ml.
5.21 Cutting template, made of a sterilizable material (stainless steel or glass) with a diameter of
3,8 cm ± 0,1 cm.
5.22 Disposable plastic bags, suitable for containing food products, to be used for storage of samples.
5.23 Tweezers, made of a material which can be sterilized.
5.24 Stainless steel cylinder, with a mass of 200 g ± 10 g and a diameter of 3,5 cm ± 0,1 cm.
5.25 Metal wire basket, for autoclaving.
5.26 Aluminium foil.
ISO 20743:2007(E)
6 Reagents and culture media
Reagents used in tests shall be of analytical quality and/or suited for microbiological purposes.
Dehydrated products available on the commercial market are recommended for use in preparing the culture
media. The manufacturer’s instructions for the preparation of these products should be strictly followed.
6.1 Water.
Water used in tests shall be analytical-grade water for microbiological media preparation which is freshly
distilled and/or ion-exchanged and/or ultra-filtered and/or filtered with RO (reverse osmosis). It shall be free
from all toxic or bacteria inhibitory substances.
6.2 Tryptone soya broth (TSB).
This solution shall be used for the resuscitation of the freeze-dried bacterial strains.
Tryptone, pancreatic digest of casein 15 g
Soya peptone, papain digest of soya 5 g
Sodium chloride (NaCl) 5 g
Water 1 000 ml (final volume)
pH after sterilization
7,2 ± 0,2
6.3 Tryptone soya agar (TSA).
Tryptone, pancreatic digest of casein 15 g
Soya peptone, papain digest of soya 5 g
Sodium chloride (NaCl) 5 g
Agar 15 g
Water 1 000 ml (final volume)
pH after sterilization 7,2 ± 0,2
6.4 Agar for transfer.
Tryptone, pancreatic digest of casein 0,75 g
Soya peptone, papain digest of soya 0,25 g
Sodium chloride (NaCl) 5 g
Agar 15 g
Water 1 000 ml (final volume)
pH after sterilization
7,2 ± 0,2
4 © ISO 2007 – All rights reserved

ISO 20743:2007(E)
6.5 Nutrient broth (NB).
Beef extract 3 g
Peptone 5 g
Water 1 000 ml (final volume)
pH after sterilization
6,9 ± 0,2
6.6 Peptone-salt solution.
Tryptone, pancreatic digest of casein 1 g
Sodium chloride (NaCl) 8,5 g
Water 1 000 ml (final volume)
pH after sterilization
6,9 ± 0,2
6.7 Physiological saline.
Sodium chloride (NaCl) 8,5 g
Water 1 000 ml (final volume)
6.8 SCDLP medium.
Peptone, digest of casein 17 g
Peptone, digest of soybean 3 g
Sodium chloride (NaCl) 5 g
Potassium dihydrogenphosphate 2,5 g
Glucose 2,5 g
Lecithin 1 g
Polysorbate 80 7 g
Water 1 000 ml (final volume)
pH after sterilization
7,2 ± 0,2
If the neutralizing power is insufficient, the content of polysorbate 80 or lecithin may be adjusted or another
neutralizing agent may be added. The use of any unspecified neutralizer shall be recorded along with the
name and concentration.
6.9 Dilution buffer for shake-out bacterial suspension.
This buffer solution consists of 0,005 mol/l sodium dihydrogenphosphate containing 0,037 % sucrose.
pH
7,2 ± 0,2
ISO 20743:2007(E)
6.10 Neutralizing solution.
The composition of the standard neutralizing solution shall be as follows.
Polysorbate 80 30 g
Egg yolk lecithin 3 g
Histidine hydrochloride 1 g
Meat or casein peptone 1 g
Sodium chloride (NaCl) 4,3 g
Monopotassium phosphate 3,6 g
Disodium phosphate dihydrate 7,2 g
Water 1 000 ml (final volume)
When sufficient neutralizing power cannot be achieved, the content of polysorbate 80 or lecithin may be
adjusted or another neutralizing agent may be added. The use of any unspecified neutralizer shall be
recorded along with the name and concentration.
6.11 Enumeration agar (EA).
Dehydrated yeast extract 2,5 g
Casein tryptone 5,0 g
Glucose 1,0 g
Agar 12 g to 18 g (depending on the gel strength of the product)
Water 1 000 ml (final volume)
pH after sterilization
7,2 ± 0,2
6.12 Agar for printing.
Agar 20 g
Water 1 000 ml (final volume)
6.13 Cryoprotective solution for bacterial species.
For freezing, a cryoprotective solution containing 150 g/l of glycerol or 100 g/l of dimethylsulfoxide shall be
used.
For solutions containing glycerol, prepare the nutritive solution and add 150 g of glycerol per litre prior to
sterilizing.
For solutions containing dimethylsulfoxide, sterilize the dimethylsulfoxide by means of a filtering system
equipped with a 0,22 µm membrane filter. Prepare the nutritive solution and, after sterilization, add 100 g of
dimethylsulfoxide per litre.
6 © ISO 2007 – All rights reserved

ISO 20743:2007(E)
NOTE Any commercially available product may be used as long as it is a cryoprotective solution or preserving
system that contains glycerol or dimethylsulfoxide and allows preservation of the strains in the same manner as the
specified solutions.
Reagents used in tests shall be of analytical quality and/or suited for microbiological purposes.
6.14 Stock solution of ATP standard reagent.
Adenosine-disodium 5’triphosphate trihydrate 59,7 mg
Water 1 000 ml (final volume)
After preparation, the solution shall be placed in a tightly sealed container and cryopreserved at a temperature
of −20 °C or lower. The solution shall be used no later than 6 months from the date of preparation.
6.15 Buffer solution for ATP luminescence reagent.
Tris(hydroxymethyl)amino methane 760 mg
Ethylenediamine disodium tetraacetate dihydrate 370 mg
Magnesium acetate tetrahydrate 800 mg
DL-dithiothreitol 8 mg
Water 250 ml (final volume)
pH
7,5 ± 0,2
This solution shall be used no later than 8 h after preparation.
6.16 ATP luminescence reagent.
Luciferase (EC: 1.13.12.7) 10 mg
D-luciferin 15 mg
Bovine serum albumin 60 mg
Buffer solution of 6.15 30 ml (final volume)
After dissolving, the reagent shall be kept at room temperature for a minimum of 15 min before use and shall
be used within 3 h of preparation.
6.17 ATP extracting reagent.
10 % aq. Benzalkonium chloride 1 ml
Water 9 ml
Use of any unspecified extracting agent and the recipe shall be recorded.
ISO 20743:2007(E)
6.18 ATP eliminating agent.
−13
An agent to reduce the ATP in nutrient broth to less than 10 mol/l within 15 min.
Apyrase (EC: 3.6.1.5) 5 international units
Adenosine deaminase (EC: 3.5.1.4) 5 international units
0,005 mol/l buffer solution of sodium dihydrogenphoshate 10 ml (final volume)
containing 0,037 % sucrose
pH
7,2 ± 0,2
Use of any unspecified eliminating agent and the recipe shall be recorded.
6.19 SCDLP or other medium for preparing ATP reference solution.
SCDLP of 6.8 or other medium 10 ml
ATP eliminating agent of 6.18 1 ml
After mixing, maintain at 30 °C to 37 °C for 1 h to prevent microbiological contamination.
Next, transfer to a hot water bath at 70 °C to 90 °C for 1 h and cool down to room temperature.
Preserve the solution under refrigeration and use within 24 h.
An ATP reference solution should be prepared if the addition of neutralizing agents causes the ATP content in
−11
the shake-out solution to exceed 10 mol/l.
7 Reference strains
7.1 Strains
The following strains shall be used in all antibacterial activity tests:
⎯ Staphylococcus aureus ATCC 6538, CIP 4.83, DSM 799, NBRC 13276 or NCIMB 9518;
⎯ Klebsiella pneumoniae ATCC 4352, CIP 104216, DSM 789, NBRC 13277 or NCIMB 10341.
NOTE ATCC is the American Type Culture Collection (USA); CIP is the Pasteur Institute Collection (France); DSM is
the German Collection of Microorganism and Cell Cultures (Germany); NBRC is the NITE Biological Resource Center
(Japan); and NCIMB is the National Collection of Industrial Bacteria (UK).
In lieu of the specified strains, equivalent bacteria strains obtained from agencies of the World Federation of
Culture Collection (WFCC) may be used by agreement between the interested parties. The strains used in the
test shall be stated in the test report together with their supply sources.
7.2 Storage of strains
7.2.1 General
The strains shall be stored in accordance with the supplier’s recommendations or EN 12353.
The identification and origin (culture collection) of the strains as well as the laboratory storage method shall be
recorded.
8 © ISO 2007 – All rights reserved

ISO 20743:2007(E)
7.2.2 Ceramic bead method
Obtain a sample of the freeze-dried bacterial strain following the recommendations supplied with the culture
and resuspend in 5 ml of TSB (6.2). Obtain a sample of the suspension and isolate it in a Petri dish (5.17)
containing TSA (6.3). Incubate the cultures for 18 h to 24 h at 37 °C ± 2 °C.
After incubation, use the culture isolated in the Petri dish to verify the purity of the strain.
After verification, prepare the stock cultures.
Sample 0,7 ml of the broth culture and spread it over the surface of the Petri dish containing the TSA.
Incubate the culture on plates for 18 h to 24 h under the conditions specified for the strain in the standard.
Add 10 ml of cryoprotective solution (6.13) to the surface of the TSA plate culture and resuspend the cells in
the solution using a sterile glass spreader. Sample the suspended cells from the surface of the agar, dilute
them in 100 ml of cryoprotective solution and incubate for 30 min at 20 °C.
Using a pipette (5.15), sample 1 ml of the suspension and transfer it to a cryogenic vial (5.16) containing the
beads (5.19). Shake the vial in order to spread the suspended cells around the beads.
⎯ Where a cryoprotective solution containing dimethylsulfoxide is used, do not let it stand longer than 1 min
at ambient temperature.
⎯ Where a cryoprotective solution containing glycerol is used, let it stand for 30 min at 20 °C.
⎯ Withdraw the excess cryoprotective solution with a sterile pipette. Place the cryogenic vials in a freezer
(5.12) set at −70 °C or lower.
−6 −7
Prepare 10 and 10 dilutions of the suspension using the serial dilution method. Take a 1,0 ml sample of
each dilution and transfer it to separate Petri dishes. Add 12 ml to 15 ml of nutritive solution, cooled down to
45 °C ± 1 °C. Incubate for 18 h to 24 h under the conditions specified for the strain. Enumerate the plate
cultures and confirm that the suspension contains less than 5 × 10 CFU/ml.
Store the cryogenic vials in a freezer at a temperature below −70 °C.
7.2.3 Glycerol suspension method
Inoculate a 15 ml culture tube containing 5 ml of appropriate medium with a freshly grown isolated colony.
Incubate at 37 °C until the culture is in the late log or stationary phase (usually 5 h to overnight).
For each strain to be stored below −70 °C, for the archives, prepare a sterile, labelled cryogenic vial. Place
225 µl of sterile 80 % glycerol in a cryogenic vial. Add 1,0 ml of the bacterial culture (frozen stock shall be
15 % glycerol). Mix well using the vortex mixer (5.4) and store in a tube at −70 °C or lower.
For each strain to be stored at −20 °C, as liquid glycerol working stock, pipette equal volumes of 80 % glycerol
and bacterial culture into a labelled polypropylene tube. Mix the contents well to avoid formation of ice crystals
that will decrease the viability of the cells. Place the tube in a freezer at −20 °C. Check the viability of the cells
after 1 week if possible.
To recover a strain from the below −70 °C glycerol stock, use a sterile toothpick to scrape pieces of the solid
substance, then streak the cells onto the appropriate medium. Do not thaw the frozen stock because each
freeze-thaw cycle will result in a 50 % loss in cell viability.
To use the −20 °C working stock, pipette 50 µl to 100 µl as inoculum for a 5 ml overnight culture.
ISO 20743:2007(E)
7.2.4 Remarks
The identity of the strain shall be verified in accordance with commonly accepted identification methods.
For each microbial strain, the following information shall be recorded:
a) the name of the national culture collection from which the freeze-dried strain was obtained;
b) the taxonomic name and reference number of the freeze-dried strain;
c) the batch number of the freeze-dried sample obtained from the culture collection;
d) the date of the resuscitated freeze-dried sample;
e) the date of prepared stock culture;
f) the stock culture laboratory code.
Details of the methods used for verifying the purity and identity of the strain and dates on which the
verifications were conducted, and the results obtained, shall also be recorded.
8 Quantitative measurement
8.1 Plate count method
8.1.1 Take 1 ml of the inoculum using a pipette (5.15), add it to a test tube containing 9,0 ml ± 0,1 ml of the
nutrient broth (6.5) or the peptone-salt solution (6.6) and shake well.
8.1.2 Take 1 ml of this solution using a new pipette, add it to a separate test tube containing 9,0 ml ± 0,1 ml
of the medium and shake well. Repeat the procedure successively and prepare a dilution series so that the
dilutions are undertaken ten times in total. Ensure that 1 ml of each dilution is pipetted into two Petri dishes
each.
8.1.3 Warm approximately 15 ml of EA (6.11) to a temperature of 45 °C to 46 °C using a water bath (5.3),
add to the dishes and mix well. Maintain at room temperature and, when the medium solidifies, turn the dishes
upside down and incubate at 37 °C ± 2 °C for 24 h to 48 h.
8.1.4 After incubation, count the number of colonies on the Petri dishes of dilution series on which 30 CFU
to 300 CFU have appeared, and obtain the bacteria concentration in the solution according to the following
formula:
c = Z ⋅ R
B
where
c is the bacteria concentration, in Colony Forming Units per millilitre (CFU/ml);
B
Z is the average value, in Colony Forming Units (CFU), in the two Petri dishes;
R is the dilution rate.
10 © ISO 2007 – All rights reserved

ISO 20743:2007(E)
8.2 Luminescence method
8.2.1 Calibration curve formula
8.2.1.1 Take the standard ATP reagent (6.14), and the physiological saline (6.7), the SCDLP medium
–8
(6.8) or another suitable medium, and prepare three separate solutions diluted by 2 × 10 mol/l,
–9 –10
2 × 10 mol/l and 2 × 10 mol/l, respectively.
8.2.1.2 Pour 0,1 ml of each solution specified above into three separate test tubes. Add 0,9 ml of the
dilution buffer (6.9) to each tube and shake amply. Pour 0,1 ml of each into three separate test tubes and
designate them as samples for measuring the diluted standard reagent.
8.2.1.3 Pour 0,1 ml of physiological saline (6.7), SCDLP (6.8) or other suitable medium, 0,8 ml of the
dilution buffer (6.9), and 0,1 ml of ATP eliminating agent (6.18) into a plastic test tube. Shake amply and pour
0,1 ml portions into three separate test tubes. Let them stand for 5 min to 30 min and designate them as
samples for measuring the blank.
8.2.1.4 Add to a test tube containing the sample for measuring the blank, 0,1 ml of ATP extracting
reagent (6.17) and shake. Add 0,1 ml of ATP luminescence reagent (6.16), shake again and immediately
apply a luminescence photometer (5.9) to determine the quantities of luminescence.
8.2.1.5 Add to the samples for measuring the diluted standard reagent, 0,1 ml portions of ATP extracting
reagent (6.17) in order, starting from the lowest concentration, and shake. Then add 0,1 ml of ATP
luminescence reagent (6.16), shake again and immediately apply a luminescence photometer (5.9) to
determine the quantities of luminescence.
8.2.1.6 Divide the ATP concentration by the average of the quantity of luminescence obtained from the
–8 –9 –10
measurement of diluted standard reagent (2 × 10 mol/l, 2 × 10 mol/
...