SIST EN ISO 846:1999

SLOVENSKI STANDARD
SIST EN ISO 846:1999
01-maj-1999
Polimerni materiali - Vrednotenje delovanja mikroorganizmov (ISO 846:1997)
Plastics - Evaluation of the action of microorganisms (ISO 846:1997)
Kunststoffe - Bestimmung der Einwirkung von Mikroorganismen auf Kunststoffe (ISO
846:1997)
Plastiques - Evaluation de l'action des micro-organismes (ISO 846:1997)
Ta slovenski standard je istoveten z: EN ISO 846:1997
ICS:
07.100.99 Drugi standardi v zvezi z Other standards related to
mikrobiologijo microbiology
83.080.01 Polimerni materiali na Plastics in general
splošno
SIST EN ISO 846:1999 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 846:1999

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SIST EN ISO 846:1999

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SIST EN ISO 846:1999

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SIST EN ISO 846:1999
IS0
INTERNATIONAL
846
STANDARD
Second edition
1997006- 15
Evaluation of the action
Plastics -
of microorganisms
Plastiques - haha tion de /‘action des micro-organismes
Reference number
IS0 846:1997(E)

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SIST EN ISO 846:1999
IS0 846: 1997(E)
Page
Contents
iv
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .*.
Introduction
1
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 Scope
2
................................................................
2 Normative references
2
..................................................................................
3 Definitions
2
Principle .
4
3
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 Apparatus and materials
7
Test specimens .
6
8
..........................................................
7 Preparation of specimens
9
Procedures .
8
14
...............................................................................
9 Assessment
16
Expression of results .
10
17
.................................................
11 Accuracy of the measurements
17
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12 Test report
18
..~............................................................................
13 Bibliography
Annexes
A Determination of the water content and water-holding capacity
19
of a soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21
B Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C Information on test fungi
0 IS0 1997
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced
or utilized in any form or by any means, electronic or mechanical, including photocopying and
microfilm, without permission in writing from the publisher.
International Organization for Standardization
Case postale 56 l CH-1211 Geneve 20 l Switzerland
Internet central @ iso.ch
x.400 c=ch; a=400net; p=iso; o=isocs; s=central
Printed in Switzerland
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SIST EN ISO 846:1999
Q IS0 IS0 846: 1997(E)
Foreword
IS0 (the International Organization for Standardization) is a worldwide
federation of national standards bodies (IS0 member bodies). The work of
preparing International Standards is normally carried out through IS0
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. IS0
collaborates closely with the International Electrotechnical Commission
(IEC) on all matters of electrotechnical standardization.
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.
International Standard IS0 846 was prepared by Technical Committee
lSO/TC 61, Plastics, Subcommittee SC 6, Ageing, chemical and environ-
mental resistance.
This second edition cancels and replaces the first edition (IS0 846:1978),
which has been technically revised.
The Plastics Project Group of the IBRG (International Biodeterioration
Research Group) carried out several interlaboratory tests between 1984
and 1990, using the 1978 edition of this standard, with the aim of checking
the reproducibility of the test results. The experience gained from these
tests has been incorporated in the present edition. In addition, a soil-burial
test method has been included in subclause 8.5, based on a specification
the Eidgenossische Materialprijfungsanstalt in St. Gallen, Switzerland.
Annex A forms an integral part of this International Standard. Annexes B
and C are for information only.
. . .
III

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Introduction
Under certain climatic and environmental conditions, microorganisms may
settle on and colonize the surface of plastics or plastics products. Their
presence and/or their metabolic products may not only damage the plastic
itself, but may also affect the serviceability of building materials and
systems containing plastic parts.
The tests and test condit :ions l specif #ied in this lnternationa ,I Standard are
empirical and cover most- but not all- poten tial applications.
For specific applications and for long -term tests, procedures
agreed upon which reflect performance under actual conditions.
The actions of microorganisms on plastics are influenced by two different
processes:
a) direct action: the deterioration of plastics which serve as a nutritive
substance for the growth of the microorganisms;
indirect action: the influence of metabolic products of the micro-
b)
or fu rther deterioration.
organisms, e.g. discolouration
This International Standard deals with both of these two processes, as well
as their combined action.
iv

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SIST EN ISO 846:1999
-
IS0 846:1997( E)
INTERNATIONAL STANDARD 0 Iso
Plastics - Evaluation of the action of microorganisms
WARNING - Handling and manipulation of microorganisms which are potentially hazardous requires
a high degree of technical competence and may be subject to current national legislation and regulations.
Only personnel trained in microbiological techniques should carry out such tests. Codes of practice for
disinfection, sterilization and personal hygiene must be strictly observed.
It is recommended that workers consult IEC 6812110:1988, appendix A “Danger to personnel”, and
General rules for microbiological
IS0 7218:1996, Microbiology of food and animal feeding stuffs -
examinations.
1 Scope
This International Standard specifies methods for determining the deterioration of plastics due to the action
of fungi and bacteria and soil microorganisms. The aim is not to determine the biodegradability of plastics.
The type and extent of deterioration may be determined by
a) visual examination
and/or
b) changes in mass
and/or
changes in other physical properties.
C)
The tests are applicable to all articles made of plastic that have an even surface and that can thus be easily
cleaned. The exceptions are porous materials, such as plastic foams.
This International Standard uses the same test fungi as IEC 68-2- 10. The IEC method, which uses so-called
“assembled specimens”, calls for inoculation of the specimens with a spore suspension, incubation of the inoculated
specimens and assessment of the fungal growth as well as any physical attack on the specimens.
The volume of testing and the test strains used will depend on the application envisaged for the plastic. These
parameters should therefore be agreed upon before the tests and should be stated in the test report.

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SIST EN ISO 846:1999
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2 Normative references
The following standards contain provisions which, through reference in this text, constitute provisions of this
International Standard. At the time of publication, the editions indicated were valid. All standards are subject to
revision, and parties to agreements based on this International Standard are encouraged to investigate the
possibility of applying the most recent editions of the standards indicated below. Members of IEC and IS0 maintain
registers of currently valid International Standards.
IS0 291 :-I), Plasfics - Standard atmospheres for conditioning and testing.
IEC 68 -2 - IO: 1988, Basic environmental testing procedures - Par? 2: Tests - Test J and guidance: Mould growth.
3 Definitions
For the purposes of this International Standard, the following definitions apply:
3.1 biodeterioration: A change in the chemical or physical properties of a material due to the action of a
microorganism.
3.2 fungistatic effect: The antimycotic effect of an antimicrobial treatment which prevents a given material from
being overgrown by fungi under moist conditions.
3.3 biodegradation: The term “biodegradation” is being discussed by TC 61/SC 5/W/G 22, Biodegradability, and
the official definition will be included here when it is available.
4 Principle
4.1 The test involves exposing test specimens of plastic to the action of selected test strains of fungi and bacteria
(or, in the case of the soil-burial test, to microbially active soil) for specified or agreed periods of time under
specified conditions of temperature and humidity.
At the end of the exposure, the test specimens are assessed before and/or after clean ing by visual examination
r other physical properties is determined.
and/or change in mass o
any
(batch I) are compared with
The results obtained with the specimens exposed to biological attack those obtained
from untreated specimens (batch 0) or sterile specimens (batch S) kept under the sa ,me conditions.
4.2 Short descriptions of the test methods used to determine the resistance of plastics to fungi (method A)
or the fungistatic effects (methods B and B’), resistance to bacteria (method C) and resistance to soil
microorganisms (method D) are given below.
4.2.1 Resistance to fungi
4.2.1.1 Method A: Fungal-growth test
Test specimens are exposed to a mixed suspension of fungus spores in the presence of an incomplete nutritive
medium (without a carbon source). The fungi can only grow at the expense of the material. If the specimens contain
no nutritive component, the fungi cannot develop mycelia and there is no deterioration of the plastic.
1) To be published. (Revision of IS0 291:1977)
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SIST EN ISO 846:1999
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Method A is suitable for the assessment of the inherent resistance of plastics to fungal attack in the absence of
other organic matter.
It is recommended that, when method A is carried out, methods B and B’ are also carried out to assist in the
interpretation of the results.
4.2.1.2 Methods B and B’: Determination of fungistatic effects
Test specimens are exposed to a mixed suspension of fungus spores in the presence of a complete medium,
i.e. with a carbon source. Even if the plastic does not contain any nutritive elements, the fungi can grow over the
specimens and their metabolic products can attack the material.
Any inhibition of the growth either on the plastic or in the growth medium (zone of inhibition) shows fungistatic
activity of the plastic or the presence of a fungicidal treatment.
In method B’, the specimens are not placed on the nutritive medium until it is completely overgrown.
Methods B and B’ are used when surface contamination is expected. In order to save time, and for a better
understanding of the phenomenon, it is recommended that the two methods are carried out simultaneously.
4.2.2 Method C: Resistance to bacteria
The action of bacteria on test specimens is assessed using a n incomplete medium. If there is no g rowth in the agar
round the specimen, then the specimen does not contain any nutritive components.
4.2.3 Method D: Resistance to microbially active soil (soil-burial test)
Test specimens are completely buried in natural soil with a known water-holding capacity and a specified moisture
content (see annex A).
The soil-burial test has been included in this International Standard because many plastics are used in permanent
contact with soil and exposed to high humidities.
4.3 Choice of properties for assessment of biodeterioration
The choice of the properties to be determined depends on the aim of the test. A visual assessme nt of biological
attack should preferably always be made as the first stage in assessing the resistance of the plastic.
The measurement of changes in mass is recommended, especially for those plastics that contain biologically
degradable substances, such as plasticizers, lubricants and stabilizers (as in plasticized PVC, for instance). The
measured loss is, in this case, often lower than the actual loss as the biologically degradable substance is only
partly utilized and the metabolic products often remain in the plastic.
When, above a .II, the surface is affected, it is recommended that determinations be made of those properties which
clearly indicate surface changes, such as surface gloss, flexural properties, impa ct resistance and hardness.
5 Apparatus and materials
5.1 For all tests
5.1 .l Incubators
That used for tests involving fungal and bacterial attack shall be capable of controlling the temperature to -t-j OC
at any temperature from 20 “C to 35 “C at a relative humidity of 90 % or greater.

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SIST EN ISO 846:1999
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That used for soil-burial tests shall be capable of controlling the temperature to f 1 “C at 29 “C at a relative humidity
of 95% or greater.
NOTE - Experience has shown that it is preferable to use two incubators: one for Petri dish tests and another for soil-burial
tests.
5.1.2 Oven, capable of controlling the temperature at 45 “C for drying test specimens and at between 103 “C and
105 “C for determining the water-holding capacity of soil.
5.1.3 Desiccator, capable of maintaining standard temperature and humidity conditions (23 OC and 50 % R.H.)
for the conditioning of test and control specimens.
51.4 Autoclave, capable of maintaining a temperature and pressure of 120 “C and 2 bar, respectively, for
sterilizing Petri dishes and soil.
5.1.5 Analytical balance, accurate to 0,l mg.
5.1.6 Laboratory centrifuge.
5.1.7 Stereoscopic microscope, magnification x 50.
5.1.8 Glass or plastic disposable Petri dishes, of suitable size for exposing test specimens.
5.1.9 Glass containers, with a volume of about 1 litre (height 16 cm; diameter 11 cm), for example preserving jars
with covers.
5.1 .lO Distilled or deionized water.
The water used for the preparation of all solutions and nutritive media and for all determinations shall be distilled
or deionized and have a conductivity of < 1 @/cm.
5.1 .ll Microbicidal solutions:
5.1.11.1 Ethanol-water mixture, in the proportions, by mass, of 70:30.
5.1 .11.2 o-Phenylphenol.
Dissolve 1 g of ephenylphenol in 50 ml of 90 % ethanol, make up to 1 000 ml with water and adjust the pH to 3,s by
adding lactic acid drop by drop.
The microbicidal solution used shall be stated in the test report.
5.2 For tests with fungi
5.2.1 Test fungi.
The test fungi shall be obtained from national culture collections The strains to be used are listed in table 1, and be
stated in the test report.
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Table 1
Name
Strain
Aspergillus niger van Tieghem
ATCC 6275
Penicillium funiculosum Thorn CMI 114933
Paecilomyces variotii Bainier
ATCC 18502
Gliocladium virens Miller et al. ATCC 9645
Chaetomium globosum Kunze: Fries
ATCC 6205
If there are technical reasons, and by agreement between the interested parties, other species may be used. In this
case, too, the strains used shall be stated in the test report.
When carrying out tests on plastics intended for use in electronic components and electronic equipment, using the
method specified in IEC 68 -2 - 10, use Aspergihs niger, Penicilium funicu/osum, Paecilomyces variotii and
Gliocladium virens from table 1 and the four strains given in table 2.
Table 2
Name Strain
Aspergillus terreus Thorn QM 82j
Aureobasidium pullulans (de Bar-y) Arnaud ATCC 9348
Penicillium ochrochloron Biourge ATCC 9112
Scopulariopsis brevicaulis (Saccardo) Bainier CMI 49528
5.2.2 Stock strains.
Culture the test fungi (5.2.1) in tubes on agar slants of the following composition:
Oatmeal
20 g
Malt extract
IQ
Agar
20 g
Water 1 000 ml
Sterilize at 120 “C + 1 “C for 20 min in an autoclave in an atmosphere saturated with water vapour.
After incubation at 29 OC + 1 OC or 24 “C + 1 “C, well sporulating cultures may then be used. They shall not be
stored for more than 4 weeks at this temperature.
Because of the possibility of genetic and physiological changes in the test fungi during culturing on artificial media,
the intervals between subculturing shall be reduced to a minimum by suitable measures (e.g. lyophilization
of cultures, storage at + 4 “C or in liquid nitrogen).
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5.2.3 Solutions and nutritive media:
5.2.3.1 Stock mineral-salt solution, of the following composition (use only chemicals of analytical grade or
equivalent purity):
NaNOs 290 g
KH2P04 097 g
K2HP04 093 g
KCI 095 g
MgS04*7H20
0’5 g
FeS04-7H20
WI g
H20 1 000 ml
Adjust the pH to 6,0 to 6,5 with sterile 0,Ol mol/l NaOH solution.
5.2.3.2 Mineral-salt/wetting-agent solution, prepared by adding to 1 litre of stock mineral-salt solution (5.2.3.1)
0,l g of a non-toxic wetting agent such as IV-methyltaurine or polyglycol ether and sterilizing in an autoclave at
120 “C + 1 “C for 20 min.
5.2.3.3 Mineral-salt/glucose solution, prepared by adding to stock mineral-salt solution (5.2.3.1) sufficient
glucose to give a concentration of 30 g/l + 1 g/l and sterilizing in an autoclave at 115 “C + 1 “C for 30 min.
5.2.3.4 Incomplete agar medium, prepared by adding to stock mineral-salt solution (5.2.3.1) sufficient agar to
give a concentration of 20 g/l. Dissolve the agar by boiling the solution whilst stirring. Sterilize in an autoclave at
120 “C + 1 “C for 20 min. Adjust the pH to 6,0 to 6,5 with sterile 0,Ol mol/l NaOH solution.
5.2.3.5 Complete agar medium, prepared by adding to the incomplete agar medium (5.2.3.4) sufficient glucose to
give a concentration of 30 g/l rt 1 g/l. Sterilize in an autoclave at 115 “C + 1 “C for 30 min. After sterilization, adjust
the pH to between 6,0 and 6,5 at 20 “C with sterile 0,Ol mol/l NaOH solution.
5.3 For tests with bacteria
5.3.1 Test bacterium Pseudomonas aeruginosa, strain NCTC 8060 or ATCC 13388.
A well-defined strain of the test bacterium shall be obtained from a national culture collection. Cultivate the test
strain on brain-heart infusion agar (5.3.2.1).
If, by agreement, additional test bacteria are used, they shall be mentioned in the test report.
5.3.2 Nutritive media and solutions
5.3.2.1 Brain-heart infusion agar.
Casein soybean peptone agar may be used as an alternative.
The medium may be obtained from commercial suppliers and shall be prepared in accordance with the
manufacturer’s instructions
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SIST EN ISO 846:1999
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5.3.2.2 Brain-heart infusion broth.
Casein soybean peptone broth may be used as an alternative.
The medium may be obtained from commercial suppliers and shall be prepared in accordance with the
manufacturer’s instructions.
5.3.2.3 Mineral-salt agar, prepared by making up a solution of the following composition:
KH2P04
097 g
K2HP04
017 g
MgS04*7H20
0’7 9
NH4N03
llg
0,005 g
NaCI
FeS04=7H20 0,002 g
0,002 g
ZnS04=7H20
MnS0407H20 0,001 g
1 000 ml
H20
and adding 20 g of agar to the solution. Sterilize in an autoclave at 120 “C + 1 “C for 20 min. Adjust the pH to 7,0
at 20 “C with 0,Ol mol/l NaOH solution.
5.3.2.4 Sterile buffer solution, pH 7,0 at 20 “C.
Prepare the following two solutions separately:
9,l g/l (solution A)
KH2P04
Na2HP04 II,9 g/l (solution B)
Mix 600 ml of solution A with 400 ml of solution B. Sterilize in an autoclave at 120 OC k 1 OC for 20 min. Adjust the
pH to 7,0 at 20 “C by adding 0,Ol mol/l NaOH solution.
5.4 For soil-burial tests
Use an activated soil with a moisture content of (60 + 5) % of the water-holding capacity of the soil (see annex A).
The water-holding capacity is the water content of a soil when it is saturated with water.
The pH of an aqueous soil extract (1 g of soil in 20 g of water) shall be between 4,0 and 7,0.
Determine the moisture content and water-holding capacity of the soil in accordance with annex A. If the moisture
content of the soil exceeds the above figure, spread it out in a thin layer under ambient laboratory conditions. Do not
heat the soil or allow it to dry out as this may affect the soil microflora. If the moisture content needs to be raised,
use an aqueous solution of 1 g of ammonium nitrate and 0,2 g of dipotassium phosphate in 1 litre of water.
6 Test specimens
6.1 Shape and dimensions
The shape and dimensions of the specimens will depend on any tests to be carried out following exposure to the
fungi, bacteria or soil.
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If it is necessary to measure changes in the thickness of the specimens, use specimens taken from the original
material. If the material is to be moulded before use, use specimens of maximum thickness 05 mm.
If changes in mass are to be measured, use square specimens of side 30 mm to 60 mm with a maximum thickness
of 2 mm. When visual examination is used to assess changes in appearance, specimen dimensions are not as
critical. However, a thickness of 05 mm to 2 mm is recommended.
Since the microorganisms may attack the surface of the plastic tested, only results using specimens of the same
dimensions may be compared.
6.2 Specimen batches and numbers in each batch
6.2.1 Specimen batches
For each sample and each test method, prepare three batches of specimens:
batch 0: control specimens, stored under standard temperature and moisture conditions;
batch I: specimens inoculated with microorganisms and incubated;
batch S: sterile specimens, stored under the same conditions as batch I.
6.2.2 Numbers in each batch
For visual examination, prepare at least five specimens for each batch, i.e. a total of at least 15 specimens per
sample and per test method.
For determination of mass changes, prepare at least six specimens for each batch, i.e. a total of at least
18 specimens per sample and per test method.
For other assessment procedures, use the number of specimens specified in the referring standard.
The test procedure for each assessment shall be carried out separately.
in
However, specime ns for determining changes mass or other physical properties also be used for visual
may
examination.
7 Preparation of specimens
7.1 Cleaning
Dip specimens for methods A and C into an ethanol-water mixture (5.1 .I 1 .I) for 1 min and dry at 45 “C for 4 h,
unless they are adversely affected by ethanol. In the latter case, store the specimens in a sterile container, handling
them with sterile forceps. Carry out all subsequent handling of the specimens using forceps to avoid contamination
by extraneous organic matter.
Do not clean specimens for methods B, B’ or D.
7.2 Labelling and storage
Store the cleaned and labelled (or marked) specimens in Petri dishes (5.1.8) at ambient temperature.
Labelling or marking may result in surface reactions by the plastic during the test. In such cases, store the
specimens separately in suitable containers (e.g. Petri dishes) and mark the Petri dishes, not the specimens, to
avoid surface reactions In all other cases, the specimens may be labelled directly using a suitable marker.
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7.3 Conditioning and weighing
Store batches of specimens used for determining change in mass at ambient temperature in a desiccator (5.1.3)
until the mass of each specimen (ml, 1-122, ma, etc.) is constant to the nearest 0,l mg (usually after 48 h). Record the
mass of each specimen. Unless otherwise agreed, specimens for visual examination and/or for determination of
changes in physical properties other than mass do not need conditioning at this stage.
It may be agreed between the interested parties to store the specimens in a desiccator (5.1.3) at 45 “C. In this case,
cool over silica gel to room temperature before use and store until constant mass is reached at 20 “C + 1 “C and
(65 + 3) % R.H. If this procedure is followed, it shall be mentioned in the test report.
8 Procedures
8.1 Test temperature
Prepare and assess specimens at standard conditions (see IS0 291) of 23 “C k 1 “C and (50 + 3) % R.H.; incubate
themat24”Ckl “Cor29”Cfl OC.
8.2 Test methods
A general scheme of the test methods described is shown in table 3.
The choice of method and of the properties to be measured depends on the material under test and the conditions
of use envisaged to it.
Table 3 - Summary of test methods
Tests Tests
Tests with fungi
with bacteria with soil
Method A B B’ C D
8.2.2 8.2.2.7 8.2.3 8.2.4
Subclause 8.2.1
Mineral-salt agar
incomplete agar Complete agar Complete agar (5.3.2.3)
Soil
medium medium None medium inoculated as
Medium used
(see 5.4)
(5.2.3.5) (5.2.3.5) specified in
(5.2.3.4)
8.2.3.5
Batch I S I S I I S I S I S
Solution
sprayed on sp-s MS-S sp-s MS-S sp-s sp-s MS-S None MS-S None MS-S
specimenl)
24”CH “Cor29”Cfl “C 29 “C + 1 “C
Incubation
conditions
4 weeks or more; > 95 % relative humidity*)
1) Sp-S = spore suspension; MS-S = microbicidal solution.
2) This humidity is reached by the agar medium in methods A, B, B’ and C. For method D, the incubator shall have
a controlled relative humidity of at least 95 %.
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8.2.1 Fungal-growth test (method A)
8.2.1.1 Filling the Petri dishes
After sterilization, pour incomplete agar medium (5.2.3.4) into sterile Petri dishes to give a depth of about 5 mm.
It solidifies on cooling.
8.2.1.2 Arrangement of test specimens
Place the specimens separately, as flat as possible, on the solidified medium, avoiding any contact between
specimens or with the walls of the Petri dishes. Divide the prepared Petri dishes randomly into two equal batches,
one labelled I, the other S.
If it is anticipated that the specimens may lift away from the medium, ballast them with weights.
8.2.1.3 Preparation of spore suspension
Produce a spore suspension from well sporulated cultures, using mineral-salt/wetting-agent solution (5.2.3.2),
as follows:
8.2.1.3.1 Harvesting the spores
Introduce into each culture tube (see 5.2.2) 5 ml of mineral-salt/wetting agent solution. Gently scrape the surface of
the sporulating culture with a sterile inoculation needle to obtain an aqueous suspension of the spores. Gently
shake the culture tube to disperse the spores in the liquid. Repeat this procedure with the same culture tube three
times. Then shake the spore suspension of each fungal culture with sterile glass beads and filter through a thin
layer of sterile cotton or glass wool to remove mycelial fragments.
8.2.1.3.2 Washing the spores by centrifugation, and preparation of working suspensions
Aseptically centrifuge the filtered spore suspension and discard the supernatant liquid. Re-suspend the residue in
25 ml of mineral-salt solution (5.2.3.1) and centrifuge again. Suspend the washed residue in 50 ml of stock mineral-
salt solution. This repeated washing of the spore suspensions is intended to guarantee that all surface-active
substances are removed which might cause stress cracking in some plastics.
Adjust the concentration to about 106 spores/ml (determined using a counting chamber or by turbimetry).
Repeat these operations with each test fungus. Blend equal volumes of five suspensions containing the same
number of spores to obtain the final mixed spore suspension ready for inoculation. Use the spore suspension within
6 h of preparation.
NOTE - When new plastics formulations are tested, the investigator may wi
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