EN 1650:2008

SLOVENSKI STANDARD
01-julij-2008
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Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation
of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in food,
industrial, domestic and institutional areas - Test method and requirements (phase 2,
step 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der fungiziden oder levuroziden Wirkung chemischer Desinfektionsmittel
und Antiseptika in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche
Einrichtungen - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour
l'évaluation de l'activité fongicide ou levuricide des antiseptiques et des désinfectants
chimiques utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans les
domaines domestiques et en collectivité - Méthode d'essai et prescriptions (phase 2,
étape 1)
Ta slovenski standard je istoveten z: EN 1650:2008
ICS:
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN 1650
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2008
ICS 71.100.35 Supersedes EN 1650:1997
English Version
Chemical disinfectants and antiseptics - Quantitative suspension
test for the evaluation of fungicidal or yeasticidal activity of
chemical disinfectants and antiseptics used in food, industrial,
domestic and institutional areas - Test method and requirements
(phase 2, step 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif Chemische Desinfektionsmittel und Antiseptika -
de suspension pour l'évaluation de l'activité fongicide ou Quantitativer Suspensionsversuch zur Bestimmung der
levuricide des antiseptiques et des désinfectants chimiques fungiziden oder levuroziden Wirkung chemischer
utilisés dans le domaine de l'agro-alimentaire, dans Desinfektionsmittel und Antiseptika in den Bereichen
l'industrie, dans les domaines domestiques et en Lebensmittel, Industrie, Haushalt und öffentliche
collectivité - Méthode d'essai et prescriptions (phase 2, Einrichtungen - Prüfverfahren und Anforderungen (Phase 2,
étape 1) Stufe 1)
This European Standard was approved by CEN on 5 April 2008.
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
© 2008 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 1650:2008: E
worldwide for CEN national Members.

Contents Page
Foreword.3
Introduction .4
1 Scope .5
2 Normative references .6
3 Terms and definitions .6
4 Requirements.6
5 Test method.7
5.1 Principle.7
5.2 Materials and reagents.7
5.3 Apparatus and glassware .11
5.4 Preparation of test organism suspensions and product test solutions .13
5.5 Procedure for assessing the fungicidal or yeasticidal activity of the product .16
5.6 Experimental data and calculation.22
5.7 Verification of methodology .25
5.8 Expression of results and precision.26
5.9 Interpretation of results - conclusion .26
5.10 Test report .27
Annex A (informative) Referenced strains in national collections.29
Annex B (informative) Examples of neutralizers of the residual antimicrobial activity of chemical
disinfectants and antiseptics and rinsing liquids .30
Annex C (informative) Graphical representations of dilution-neutralization method and
membrane filtration method .32
Annex D (informative) Example of a typical test report.36
Annex E (informative) Precision of the test result .42
Bibliography .45

Foreword
This document (EN 1650:2008) has been prepared by Technical Committee CEN/TC 216 “Chemical
disinfectants and antiseptics”, the secretariat of which is held by AFNOR.
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 November 2008, and conflicting national standards shall be withdrawn
at the latest by November 2008.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 1650:1997.
This European Standard was revised to include the results of a collaborative trial (ANDISTAND), to correct
obvious errors and ambiguities, to harmonize the structure and wording with other quantitative suspension
tests of CEN/TC 216 (existing or in preparation), and to improve the readability of the standard and thereby
make it more understandable.
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 the United Kingdom.

Introduction
This European Standard specifies a suspension test for establishing whether a chemical disinfectant or
antiseptic has or does not have a fungicidal or yeasticidal activity in the fields described in the scope.
This laboratory test takes into account practical conditions of application of the product including contact time,
temperature, test organisms and interfering substance, i.e. conditions which may influence its action in
practical situations.
The conditions are intended to cover general purposes and to allow reference between laboratories and
product types. Each utilization concentration of the chemical disinfectant or antiseptic found by this test
corresponds to defined experimental conditions. However, for some applications the recommendations of use
of a product may differ and therefore additional test conditions should be used.
1 Scope
This document specifies a test method and the minimum requirements for fungicidal or yeasticidal activity of
chemical disinfectant and antiseptic products that form a homogeneous, physically stable preparation when
diluted with hard water or - in the case of ready-to-use-products - with water. Products can only be tested at a
concentration of 80 % or less as some dilution is always produced by adding the test organisms and
interfering substance.
This document applies to products that are used in food, industrial, domestic and institutional areas excluding
areas and situations where disinfection is medically indicated and excluding products used on living tissues
except those for hand hygiene in the above considered areas. The following areas are at least included:
a) processing, distribution and retailing of:
1) food of animal origin: 2) food of vegetable origin:
 milk and milk products;  beverages;
 meat and meat products;  fruits, vegetables and derivatives
(including sugar, distillery .);
 fish, seafood, and related products;  flour, milling and baking;
 eggs and egg products;  animal feeds;
 animal feeds;  etc.
 etc.
b) institutional and domestic areas:
 catering establishments;
 public areas;
 public transports;
 schools;
 nurseries;
 shops;
 sports rooms;
 waste containers (bins .);
 hotels;
 dwellings;
 clinically non-sensitive areas of hospitals;
 offices;
 etc.
c) other industrial areas:
 packaging material;
 biotechnology (yeast, proteins, enzymes, .);
 pharmaceutical;
 cosmetics and toiletries;
 textiles;
 space industry, computer industry;
 etc.
EN 14885 specifies in detail the relationship of the various tests to one another and to “use
recommendations”.
NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances
under the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2 step 1 test.
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.
EN 12353, Chemical disinfectants and antiseptics – Preservation of test organisms used for the determination
of bactericidal, mycobactericidal, sporicidal and fungicidal activity
EN 14885, Chemical disinfectants and antiseptics – Application of European Standards for chemical
disinfectants and antiseptics
ISO 4793, Laboratory sintered (fritted) filters – Porosity grading, classification and designation
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 14885 apply.
4 Requirements
The product shall demonstrate a reduction of at least a 4 decimal log (lg) when diluted with hard water
(5.2.2.7) or - in the case of ready-to-use products - with water (5.2.2.2) and tested in accordance with Clause
5 under simulated clean conditions (0,3 g/l bovine albumin solution - 5.2.2.8.2) or simulated dirty conditions
(3 g/l bovine albumin solution - 5.2.2.8.3) according to its practical applications and under the other obligatory
test conditions (one or two selected test organisms, 20 °C, 15 min).
The fungicidal activity shall be evaluated using the following two test organisms:
 Candida albicans (vegetative cells);
 Aspergillus niger (spores).
The yeasticidal activity shall be evaluated using the following test organism:
 Candida albicans (vegetative cells);
Where indicated, additional specific fungicidal or yeasticidal activity shall be determined applying other contact
times, temperatures, interfering substances and test organisms (in accordance with 5.2.1, 5.2.2.8 and 5.5.1.1)
in order to take into account intended specific use conditions.
NOTE For these additional conditions, the concentration defined as a result can be lower than the one obtained
under the obligatory test conditions.
5 Test method
5.1 Principle
5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready to use
products) is added to a test suspension of fungi (yeast cells or mould spores) in a solution of an interfering
substance. The mixture is maintained at (20 ± 1) °C for 15 min ± 10 s (obligatory test conditions). At the end of
this contact time, an aliquot is taken, and the fungicidal and/or the fungistatic activity in this portion is
immediately neutralized or suppressed by a validated method. The method of choice is dilution-neutralization.
If a suitable neutralizer cannot be found, membrane filtration is used. The numbers of surviving fungi in each
sample are determined and the reduction is calculated.
5.1.2 The test is performed using the vegetative cells of Candida albicans and the spores of Aspergillus
niger (fungicidal activity) or only the vegetative cells of Candida albicans (yeasticidal activity) as test
organisms (obligatory test conditions).
5.1.3 Additional and optional contact times and temperatures are specified. Additional test organisms can
be used.
5.2 Materials and reagents
5.2.1 Test organisms
1)
The fungicidal activity shall be evaluated using the following strains as test organisms:
ATCC 10231
 Candida albicans
 Aspergillus niger ATCC 16404
The yeasticidal activity shall be evaluated using only Candida albicans.
NOTE See annex A for strain references in some other culture collections.
The required incubation temperature for these test organisms is (30 ± 1) °C (5.3.2.3).
If required for specific applications, additional strains may be chosen from, e.g. for breweries:

1)
The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection (ATCC).
This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of
the product named.
DSM 1333
 Saccharomyces cerevisiae
DSM 70487
 Saccharomyces cerevisiae var. diastaticus
If additional test organisms are used, they shall be incubated under optimum growth conditions (temperature,
time, atmosphere, media) noted in the test report. If the additional test organisms selected do not correspond
to the specified strains, their suitability for supplying the required inocula shall be verified. If these additional
test organisms are not classified at a reference centre, their identification characteristics shall be stated. In
addition, they shall be held by the testing laboratory or national culture collection under a reference for five
years.
5.2.2 Culture media and reagents
5.2.2.1 General
All weights of chemical substances given in this standard refer to the anhydrous salts. Hydrated forms may be
used as an alternative, but the weights required shall be adjusted to allow for consequent molecular weight
differences.
The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free
from substances that are toxic or inhibitory to the test organisms.
NOTE 1 To improve reproducibility, it is recommended that commercially available dehydrated material is used for the
preparation of culture media. The manufacturer's instructions relating to the preparation of these products should be
rigorously followed.
NOTE 2 For each culture medium and reagent, a limitation for use should be fixed.
5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water.
Sterilize in the autoclave [5.3.2.1a)].
NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently
sterilized.
NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) can
be used.
NOTE 3 See 5.2.2.7 for the procedure to prepare hard water.
5.2.2.3 Malt extract agar (MEA)
Malt extract agar, consisting of:
Malt extract       30,0 g
Soya peptone, papaic digest of soybean meal        3,0 g
Agar      15,0 g
Water (5.2.2.2)   to 1 000,0 ml

Sterilize in the autoclave [5.3.2.1a)]. After sterilization, the pH of the medium shall be equivalent to 5,6 ± 0,2
when measured at (20 ± 1) °C.
NOTE In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it may be necessary to add
neutralizer to the MEA. Annex B gives guidance on the neutralizers that may be used.
5.2.2.4 Diluent
Tryptone sodium chloride solution, consisting of:
Tryptone, pancreatic digest of casein        1,0 g
Sodium chloride (NaCl)        8,5 g
Water (5.2.2.2)    to 1 000,0 ml

Sterilize in the autoclave [5.3.2.1a)]. After sterilization, the pH of the diluent shall be equivalent to 7,0 ± 0,2
when measured at (20 ± 1) °C.
5.2.2.5 Neutralizer
The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.2. It
shall be sterile.
NOTE Information on neutralizers that have been found to be suitable for some categories of products is given in
Annex B.
5.2.2.6 Rinsing liquid (for membrane filtration)
The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.3.
It shall be sterile, compatible with the filter membrane and capable of filtration through the filter membrane
under the test conditions described in 5.5.3.
NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is given in
Annex B.
5.2.2.7 Hard water for dilution of products
For the preparation of 1 000 ml of hard water, the procedure is as follows:
 prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride (CaCl ) in
water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the autoclave
[5.3.2.1a)]. Autoclaving - if used - may cause a loss of liquid. In this case make up to 1 000 ml with water
(5.2.2.2) under aseptic conditions. Store the solution in the refrigerator (5.3.2.8) for no longer than one
month;
 prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (5.2.2.2) and dilute to
1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8) for no
longer than one week;
 place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) and add 6,0 ml (5.3.2.9)
of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The pH of the hard
water shall be 7,0 ± 0,2, when measured at (20 ± 1)°C (5.3.2.4). If necessary, adjust the pH by using a
solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l
(about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water produces a
different final water hardness in each test tube. In any case the final hardness is lower than 300 mg/l of calcium carbonate
(CaCO ) in the test tube.
5.2.2.8 Interfering substance
5.2.2.8.1 General
The interfering substance shall be chosen according to the conditions of use laid down for the product.
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test.
The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g. mineral
substances, protein, carbohydrates, lipids and detergents) shall be defined.
NOTE The term “interfering substance” is used even if it contains more than one substance.
5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration)
Dissolve 0,3 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (5.2.2.2).
Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month.
The final concentration of bovine albumin in the test procedure (5.5) is 0,3 g/l.
5.2.2.8.3 Dirty conditions (bovine albumin solution – high concentration)
Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (5.2.2.2).
Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month.
The final concentration of bovine albumin in the test procedure (5.5) is 3,0 g/l.
5.2.2.8.4 Milk (dairies .)
Skimmed milk, guaranteed free of antibiotics and additives and reconstituted at a rate of 100 g powder per
litre of water (5.2.2.2), shall be prepared as follows :
 prepare a solution of 100 g milk-powder in 1 000 ml water (5.2.2.2). Heat for 30 min at (105 ± 3) °C [or
5 min at (121 ± 3 °C)].
The final concentration of reconstituted milk in the test procedure (5.5) is 10,0 g/l of reconstituted milk.
5.2.2.8.5 Yeast extract (breweries .)
Dehydrated yeast extract for bacteriology, shall be prepared as follows :
 prepare a 100 g/l solution in water (5.2.2.2), adjust to pH 7,0 ± 0,2 with sodium hydroxide (NaOH);
 sterilize in the autoclave [5.3.2.1a)].
The final concentration of yeast extract in the test procedure (5.5) is 10,0 g/l.
5.2.2.8.6 Sucrose (beverage, soft drink industries)
Prepare a 100 g/l solution of sucrose in water (5.2.2.2), sterilize by membrane filtration (5.3.2.7).
The final concentration of sucrose in the test procedure (5.5) is 10,0 g/l.
5.2.2.8.7 pH 5,0 and pH 9,0 buffer solutions (cleaning in place .)
The buffer solution used shall be described in the test report and pH values shall be recorded. The final pH in
the test tubes (together with test organisms and product) shall be controlled and found equal to 5,0 ± 0,2 or
9,0 ± 0,2.
5.2.2.8.8 Sodium dodecyl sulphate (cosmetic area .)
Prepare a 50 g/l solution of sodium dodecyl sulphate (C H NaO S) in water (5.2.2.2). Sterilize in the
12 25 4
autoclave [5.3.2.1a)].
The final concentration of sodium dodecyl sulphate in the test procedure (5.5) is 5,0 g/l.
5.3 Apparatus and glassware
5.3.1 General
Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and
reagents or the sample, except those which are supplied sterile, by one of the following methods :
a) by moist heat, in the autoclave [5.3.2.1a)]
b) by dry heat, in the hot air oven [5.3.2.1b)]
2)
5.3.2 Usual microbiological laboratory equipment and in particular, the following:
5.3.2.1 Apparatus for sterilization:
+ 3
a) for moist heat sterilization, an autoclave capable of being maintained at (121 )°C for a minimum
holding time of 15 min;
+5
b) for dry heat sterilization, a hot air oven capable of being maintained at (180 )°C for a minimum holding
+5 +5
time of 30 min, at (170 ) °C for a minimum holding time of 1 h or at (160 )°C for a minimum holding
0 0
time of 2 h.
5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, at (45 ± 1) °C (to maintain melted MEA in
case of pour plate technique) and at additional test temperatures ± 1 °C (5.5.1).
5.3.2.3 Incubator, capable of being controlled at (30 ± 1) °C.
5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C.
NOTE A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media
(5.2.2.3).
5.3.2.5 Stopwatch.
5.3.2.6 Shaker
® 3)
a) Electromechanical agitator, e.g. Vortex mixer .
b) Mechanical shaker
5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances to be
filtered.
The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with filters of
diameter 47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.7), bovine albumin
(5.2.2.8.2 and 5.2.2.8.3) and sucrose (5.2.2.8.6), and if the membrane filtration method is used (5.5.3).
The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the
micro-organisms over the membrane and to prevent overlong filtration, the device shall be set so as to obtain
the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.9 Graduated pipettes, of nominal capacities 10 ml and 1 ml and 0,1 ml, or calibrated automatic
pipettes.
5.3.2.10 Petri dishes, (plates) of size 90 mm to 100 mm.
5.3.2.11 Glass beads, 3 mm to 4 mm in diameter.
5.3.2.12 Volumetric flasks.
2)
Disposable sterile equipment is an acceptable alternative to reusable glassware.
3) ®
Vortex is an example of a suitable product available commercially. This information is given for the convenience of
users of this standard and does not constitute an endorsement by CEN of this product.
5.3.2.13 Fritted filter, with porosity of 40 µm to 100 µm according to ISO 4793.
5.3.2.14 Centrifuge, (2 000 g ).
N
5.3.2.15 Roux bottles or similar flasks.
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspensions (test and validation suspension)
5.4.1.1 General
For each test organism, two different suspensions have to be prepared: the “test suspension” to perform the
test and the “validation suspension” to perform the controls and method validation.
5.4.1.2 Preservation and stock cultures of test organisms
The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353.
5.4.1.3 Working culture of test organisms
5.4.1.3.1 Candida albicans (yeast)
In order to prepare the working culture of Candida albicans (5.2.1), prepare a subculture from the stock culture
(5.4.1.2) by streaking onto MEA (5.2.2.3) slopes or plates (5.3.2.10) and incubate (5.3.2.3). After 42 h to 48 h,
prepare a second subculture from the first subculture in the same way and incubate for 42 h to 48 h. From this
second subculture, a third subculture may be produced in the same way. The second and (if produced) third
subcultures are the working cultures.
If it is not possible to prepare the second subculture on a particular day, a 72 h subculture may be used for
subsequent subculturing, provided that the subculture has been kept in the incubator (5.3.2.3) during the 72 h
period.
Never produce and use a fourth subculture.
5.4.1.3.2 Aspergillus niger (mould)
For Aspergillus niger (5.2.1), use only the first subculture grown on MEA (5.2.2.3) in Roux bottles (5.3.2.15)
and incubate for 7 days to 9 days. No further subculturing is needed.
5.4.1.3.3 Other test organisms (yeasts or moulds)
For additional test organisms, any departure from this method of culturing the yeast or the mould or of
preparing the suspensions shall be noted, giving the reasons in the test report.
5.4.1.4 Test suspension (“N”)
5.4.1.4.1 Candida albicans
The procedure for preparing the Candida albicans test suspension is as follows.
a) Take 10 ml of diluent (5.2.2.4) and place in a 100 ml flask with 5 g of glass beads (5.3.2.11). Take the
working culture (5.4.1.3.1) and transfer loopfuls of the cells into the diluent (5.2.2.4). The cells should be
suspended in the diluent by rubbing the loop against the wet wall of the flask to dislodge the cells before
immersing in the diluent. Shake the flask for 3 min using a mechanical shaker [5.3.2.6b)]. Aspirate the
suspension from the glass beads and transfer to another tube.
7 7
4)
b) Adjust the number of cells in the suspension to 1,5 x 10 cfu/ml to 5,0 x 10 cfu/ml using diluent
(5.2.2.4), estimating the number of cfu by any suitable means. Maintain this test suspension in the water
bath at the test temperature θ [5.5.1.1a)] and use within 2 h.
NOTE The use of a spectrophotometer for adjusting the number of cells is highly recommended (approximately
620 nm wavelength — cuvette 10 mm path length). Each laboratory should therefore produce calibration data for
each test organism knowing that suitable values of optical density are generally found between 0,200 and 0,350. A
colorimeter is a suitable alternative.
-5 -6
c) For counting, prepare 10 and 10 dilutions of the test suspension using diluent (5.2.2.4). Mix [5.3.2.6a)].
Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or the spread
plate technique.
1) When using the pour plate technique, transfer each 1,0 ml sample into separate Petri dishes and add
15 ml to 20 ml melted MEA (5.2.2.3), cooled to (45 ± 1) °C.
2) When using the spread plate technique, spread each 1,0 ml sample — divided into portions of
approximately equal size — on an appropriate number (at least two) of surface dried plates
containing MEA (5.2.2.3).
For incubation and counting see 5.4.1.6.
5.4.1.4.2 Aspergillus niger
The procedure for preparing the Aspergillus niger test suspension is as follows.
a) Take the working culture (5.4.1.3.2) and suspend the spores in 10 ml of sterile 0,05 % (w/v) polysorbate
80 solution in water (5.2.2.2). Using a glass rod or spatula, detach the conidiospores from the culture
surface. Transfer the suspension into a flask and gently shake by hand for one minute together with 5 g of
glass beads (5.3.2.11). Filter the suspension through a fritted filter (5.3.2.13).
b) Carry out a microscopic examination under x 400 magnification immediately after the preparation and just
before the test, to show the absence of mycelia fragments and spore germination (check at least ten
fields of view for absence of both).
If germinated spores are present, discard the suspension.
If mycelia are present, set up a washing process (centrifugation) as follows. Transfer the filtered
suspension to centrifuge tubes. The filtered suspension is centrifuged (5.3.2.14) at 2 000 g for 20 min.
N
The conidiospores are washed at least twice by resuspension in diluent (5.2.2.4) and subsequent
centrifugation. If mycelia are still present, repeat the washing process.

7 7
c) Adjust the number of spores in the suspension to 1,5 x 10 cfu/ml to 5,0 x 10 cfu/ml using the diluent
(5.2.2.4), estimating the number of cfu by any suitable means. Use the suspension within 4 h. It can be
stored up to 2 days in the refrigerator and shall then be checked just before the test for absence of
germinated spores [5.4.1.4.2b)] In any case, adjust the temperature according to 5.5.1.4 only immediately
before the start of the test (5.5.2 or 5.5.3).
NOTE The use of a cell counting device for adjusting the number of cells is highly recommended. When using a
suitable counting chamber, follow the instructions explicitly

4)
cfu/ml = colony-forming unit(s) per millilitre.
Each laboratory should therefore produce calibration data to establish the relationship between the
counts obtained using the counting device and the counts (5.4.1.6) obtained by the pour plate or the
spread plate technique. Experienced laboratories found a better fit to the required number of spores when
the spore suspension count in the device was 10 % to 50 % higher than the number aimed at.

-5 -6
d) For counting, prepare 10 and 10 dilutions of the test suspension using diluent (5.2.2.4). Mix [5.3.2.6a)].
Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or the spread
plate technique.
1) When using the pour plate technique, transfer about half of each 1,0 ml sample into separate Petri
dishes (i.e. in duplicate = four plates) and add 15 ml to 20 ml of melted MEA (5.2.2.3), cooled to
(45 ± 1) °C.
2) When using the spread plate technique, spread about one quarter of each 1,0 ml sample on an
appropriate number (at least four) of surface dried plates containing MEA (5.2.2.3) (i.e. in duplicate –
at least eight plates).
For incubation and counting see 5.4.1.6.
5.4.1.5 Validation suspension (“Nv”)
a) To prepare the validation suspension, dilute the test suspension (5.4.1.4.1 and 5.4.1.4.2) with the diluent
2 3
(5.2.2.4) to obtain the fungal count of 3,0 x 10 cfu/ml to 1,6 x 10 cfu/ml [about one-fourth (1 + 3) of the
-4
10 dilution].
-1
b) For counting, prepare a 10 dilution with diluent (5.2.2.4). Mix [(5.3.2.6a)]. Take a sample of 1,0 ml in
duplicate and inoculate using the pour plate or the spread plate technique [with Candida albicans,
5.4.1.4.1c); with Aspergillus niger, 5.4.1.4.2d)].
c) For incubation and counting, see 5.4.1.6.
5.4.1.6 Incubation and counting of the test and the validation suspensions
For incubation and counting of the test and the validation suspensions, the procedure is as follows.
a) Incubate (5.3.2.3) the plates for 42 h to 48 h. Discard any plates that are not countable for any reason.
Count the cfu on the plates to determine the total number of cfu.
Only for Aspergillus niger incubate the plates for a further 20 h to 24 h and - if the number of colonies has
increased - for a third additional period of 20 h to 24 h. Do not recount plates that no longer show well-
separated colonies. Recount the remaining plates. If the number has increased, use only the higher
number for further evaluation.
b) Note for each plate the exact number of colonies, but record ">165" (for moulds) or ">330" (for yeasts) for
any counts higher than 165 and 330 respectively and determine the Vc values according to 5.6.2.2.
c) Calculate the numbers of cfu/ml in the test suspension “N” and in the validation suspension “Nv” using the
methods given in 5.6.2.3 and 5.6.2.5. Verify according to 5.7.
5.4.2 Product test solutions
The concentration of a product test solution shall be 1,25 times the desired test concentration because it is
diluted to 80 % during the test and the method validation (5.5.2 or 5.5.3). Product test solutions shall be
prepared in hard water (5.2.2.7) at minimum three different concentrations to include one concentration in the
active range and one concentration in the non-active range (5.8.2). The product as received may be used as
one of the product test solutions, in this case the highest tested concentration is 80%.
Dilutions of ready-to-use products, i.e. products that are not diluted when applied, shall be prepared in water
(5.2.2.2).
For solid products, dissolve the product as received by weighing at least 1,0 g ± 10 mg of the product in a
volumetric flask and filling up with hard water (5.2.2.7). Subsequent dilutions (lower concentrations) shall be
prepared in volumetric flasks (5.3.2.12) on a volume/volume basis in hard water (5.2.2.7).
For liquid products, dilutions of the product shall be prepared with hard water (5.2.2.7) on a volume/volume
basis using volumetric flasks (5.3.2.12).
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogeneous preparation that is stable during the whole procedure. If during the procedure a
visible inhomogeneity appears due to the formation of a precipitate or flocculant (for example, through the
addition of the interfering substance), it shall be recorded in the test report.
NOTE Counting micro-organisms embedded in a precipitate or flocculant is difficult and unreliable.
The concentration of the product stated in the test report shall be the desired test concentration. Record the
test concentration in terms of mass per volume or volume per volume and details of the product sample as
received.
5.5 Procedure for assessing the fungicidal or yeasticidal activity of the product
5.5.1 General
5.5.1.1 Experimental conditions (obligatory and additional)
Besides the obligatory temperature, contact time, interfering substance and test organisms additional
experimental conditions (including test organisms) may be selected according to the practical use considered
for the product (Clause 4) as follows:
a) temperature θ (in °C):
 the obligatory temperature to be tested is θ = 20 °C;
 additional temperatures may be chosen from 4 °C, 10 °C or 40 °C;
 the allowed deviation for each chosen temperature is ± 1 °C;
b) contact time t (in min):
 the obligatory contact time to be tested is t = 15 min;
 additional contact times may be chosen from 1 min, 5 min, 30 min or 60 min;
 the allowed deviation for each chosen contact time is ± 10 s (except for 1 min: ± 5 s);
c) interfering substance:
 the obligatory interfering substance to be tested is 0,3 g/l bovine albumin (5.2.2.8.2) for clean
conditions or 3 g/l bovine albumin (5.2.2.8.3) for dirty conditions according to practical applications;
 additional interfering substances as given in 5.2.2.8 shall be chosen according to the field of
application specified for the product;
d) test organisms (5.2.1):
 the obligatory test organisms for testing fungicidal activity are Candida albicans and Aspergillus
niger;
 the obligatory test organism for testing yeasticidal activity is Candida albicans.
Additional test organisms may be tested.
5.5.1.2 Choice of test method (dilution-neutralization or membrane filtration)
The method of choice is the dilution-neutralization method (5.5.2). To determine a suitable neutralizer, carry
out the validation of the dilution neutralization method (5.5.2.3, 5.5.2.4 and 5.5.2.5 in connection with 5.5.2.6)
using a neutralizer, chosen according to laboratory experience and published data.
If this neutralizer is not valid, repeat the validation test using an alternative neutralizer taking into account the
information given in Annex B.
If both neutralizers are found to be invalid, the membrane filtration method (5.5.3) may be used.
NOTE In special circumstances it may be necessary to add neutralizer to MEA (5.2.2.3).
5.5.1.3 General instructions for validation and control procedures
The neutralization and/or removal of the fungicidal and/or fungistatic activity of the product shall be controlled
and validated - only for the highest product test concentration - for each of the used test organisms and for
each experimental condition (interfering substance, temperature, contact time). These procedures
(experimental condition control, neutralizer or filtration control and method validation) shall be performed at
the same time with the test and with the same neutralizer – or rinsing liquid – used in the test.
In the case of ready-to-use-products use water (5.2.2.2) instead of hard water.
If because of problems with neutralization, a neutralizer has been added to MEA (5.5.1.2) used for the
validation and control procedures the MEA used for the test shall contain the same amount of this neutralizer
as well.
5.5.1.4 Equilibration of temperature
Prior to testing, equilibrate all reagents (product test solutions (5.4.2), test suspension (5.4.1.4), validation
suspension (5.4.1.5), diluent (5.2.2.4), hard water (5.2.2.7) and interfering substance (5.2.2.8) to the test
temperature θ [5.5.1.1a)] using the water bath (5.3.2.2) controlled at θ. Check that the temperature of the
reagents is stabilized at θ.
The neutralizer (5.2.2.5) or the rinsing liquid (5.2.2.6) and water (5.2.2.2) shall be equilibrated at a
temperature of (20 ± 1) °C.
In the case of ready-to-use-products, water (5.2.2.2) shall be additionally equilibrated to θ .
5.5.1.5 Precautions for manipulation of test organisms
Do not touch the upper part of the test tube sides when adding the test or the validation suspensions (5.4.1).
5)
5.5.2 Dilution-neutralization method

5)
For a graphical representation of this method see C.1.
5.5.2.1 General
The test and the control and validation procedures (5.5.2.2 through 5.5.2.5) shall be carried out in parallel and
separately for each experimental condition (5.5.1.1).
5.5.2.2 Test "Na" – determination of fungicidal or yeasticidal concentrations
The procedure for determining fungicidal or yeasticidal concentrations is as follows.
a) Pipette 1,0 ml of the interfering substance (5.2.2.8) into a tube. Add 1,0 ml of the test suspension
(5.4.1.4). Start the stopwatch (5.3.2.5) immediately, mix [5.3.2.6a)] and place the tube in a water bath
controlled at the chosen test temperature θ [5.5.1.1a)] for 2 min ± 10 s.
At the end of this time, add 8,0 ml of one of the product test solutions (5.4.2). Restart the stopwatch at the
beginning of the addition. Mix [5.3.2.6a)] and place the tube in a water bath controlled at θ for the chosen
contact time t [5.5.1.1b)]. Just before the end of t, mix [5.3.2.6a)] again.
b) At the end of t, take a 1,0 ml sample of the test mixture "Na" and transfer into a tube containing 8,0 ml
neutralizer (5.2.2.5) and 1,0 ml water (5.2.2.2). Mix [5.3.2.6a)] and place in a water bath controlled at
(20 ± 1)°C. After a neutralization time of 5 min ± 10 s, mix and immediately take a sample of 1,0 ml of the
neutralized test mixture "Na" (containing neutralizer, product test solution, interfering substance and test
suspension) in duplicate and inoculate using the pour plate or spread plate technique.
1) When using the pour plate technique, pipette each 1,0 ml sample into separate Petri dishes and add
15 ml to 20 ml of melted MEA (5.2.2.3), cooled to (45 ± 1) °C.
2) When using the spread plate technique, spread each 1,0 ml sample – divided into portions of
approximately equal size – on an appropriate number (at least two) of surface dried pl
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