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Ref. No. : ISO/R 846-1968 (E)
IS0
I NTERN AT1 ON A L O RG A N 1Z ATi O N FOR STAN DA RDl ZATl ON
IS0 RECOMMENDATION
R 846
P LAST1 CS
RECOMMENDED PRACTICE FOR THE EVALUATION
OF THE RESISTANCE OF PLASTICS TO FUNGI
BY VISUAL EXAMINATION
1st EDITION
October 1968
COPYRIGHT RESERVED
The copyright of IS0 Recommendations and IS0 Standards
belongs to IS0 Member Bodies. Reproduction of these
documents, in any country, may be authorized therefore only
by the national standards organization of that country, being
a member of ISO.
For each individual country the only valid standard is the national standard of that country.
Printed in Switzerland
Also issued in French and Russian. Copies to be obtained through the national standards organizations.
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BRIEF HISTORY
The IS0 Recommendation R 846, Plastics - Recommended practice for the evaluation of the
resistance of plastics to fungi bj? visual examination, was drawn up by Technical Committee
ISO/TC 61, Plastics, the Secretariat of which is held by the United States of America Standards
Institute (USASI).
Work on this question by the Technical Committee began in 1957 and led, in 1964, to the
adoption of a Draft IS0 Recommendation.
In April 1965, this Draft IS0 Recommendation (No. 813) was circulated to all the IS0
Member Bodies for enquiry. It was approved, subject to a few modifications of an editorial nature, by
the following Member Bodies :
Argentina Greece Poland
Australia Hungary Portugal
Austria India Romania
Belgium Israel Switzerland
Brazil Italy U.A.R.
Canada Japan United Kingdom
Czechoslovakia Netherlands U.S.A.
France New Zealand U.S.S.R.
One Member Body opposed the approval of the Draft :
Sweden
The Draft IS0 Recommendation was then submitted by correspondence to the IS0 Council.
which decided, in October 1968. to accept it as an IS0 RECOMMENDATION.
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ISO/R 846-1968 (I
IS0 Recommendation R 846 Octo ber 1968
P LAST1 CS
RECOMMENDED PRACTICE FOR THE EVALUATION
OF THE RESISTANCE OF PLASTICS TO FUNGI
BY VISUAL EXAMINATION
INTRODUCTION
Micro-organisms vegetate on plastics containing nutritive substances, necessary for their growth,
which are either contained in the plastics as components or are deposited subsequently thereon either
in processing or in use.
Thus, on material which is itself fungus-resistant, fungi can vegetate only in the presence of impurities.
In this case, the fungus may affect the plastics by its metabolic products influencing indirectly the
properties of the plastics.
1. SCOPE
1.1 This IS0 Recommendation describes the procedure for the evaluation of the resistanceof
plastics to attack by fungi when inoculated by these organisms.
This procedure provides for visual observation as to whether or not plastics are subject to
fungal growth but it does not provide data about changes of chemical and physical properties
caused by micro-organisms.
1.2 According to the end application of a material, certain requirements, such as water resistance,
resistance to washing, resistance to weathering, or heat stability may have necessitated certain
treatments. Therefore it should be stated explicitly in what condition the test material is being
tested, i.e., its method of pre-treatment should be indicated.
NOTE, - Since the procedure involves handling and working with fungi, it is recommended that personnel
trained in microbiology should perform the part of the procedure involving handling of organisms and
inoculated specimens.
1.3 Two methods of test are described in this IS0 Recommendation : Method A and Method B.
1.3.1 Method A determines whether the plastics under test are inert or serve as a nutritive
medium for the growth of microorganisms. The resistance of a material is judged visually
according to the degree of fungal growth on its surface.
1.3.2 Method B determines the degree of fungal growth on the surface of the material when it is
enriched by a nutritive medium. The evaluation is made visually. This method permits the
determination of the fungitoxic properties of plastics.
1.4 If the conditions of use in service of the material are taken into consideration, Method A may be
considered sufficient; if not, the test is then continued according to Method B.
1.5 For example, if plastics are used under conditions which exclude surface contamination by
organic substances, Method A is sufficient. However, materials used under conditions involving
strong surface contamination should be tested according to Method B.
1.6 In order to save time, it is recommended that the two test Methods (A and B) should be
started simultaneously.
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ISO/R 846-1968 (I
2. METHOD A
2.1 scope
Method A is intended to determine the ability of plastics themselves to support the growth of
fungi, i.e. to serve as a nutritive medium.
For this reason, the test specimens are placed on a mineral-salts agar medium (2.3.1.3) and
inoculated with the spore suspension of the fungi in an aqueous mineral-salts solution (2.3.1.4)
in order to provide for conditions under which the plastics are a nutritive medium for fungi.
2.2 Test specimens
2.2.1 The size of the test specimens is optional, but not less than three replicate test specimens
should be used.
NOTE. - Recommended dimensions of the test specimens are as follows :
30 to 50 mm square pieces, with a maximum thickness of 4 mm
2.2.2 The specimens are tested under optimum conditions for fungal growth, i.e., at 30 * 2 "Cand
relative humidity.
at 95 to 100
2.2.3 The specimens are tested as delivered, without cleaning.
NOTE. - In order to make sure that secondary contamination with nutrient substances does not
interfere with the test, the specimens may be cleaned according to section A.5 of the Annex.When
cleaned test specimens are used, this fact should be mentioned in the test report, under section 4.
Preparation of fungal species for test
2.3
2.3.1 Num'tive media. The following nutritive media are suitable for the culturing of fungi.
2.3.1.1 Modified Czapek-Dox agar, containing saccharose.
NaNO, .
2 g
KH,PO, . 0.7 g
K,HPO, . 0.3 g
KCl . 0.5 g
MgSO, .7 H,O . 0.5 g
FeSO, -7 H, O . 0.01 g
Saccharose .
30 g
Distilled water . 1 O00 ml
Agar .
20 g
2.3.1.2 Modified Czapek-Dox agar, containing cellulose, In the nutritive medium mentioned
in clause 2.3.1.1 above, saccharose is replaced by cellulose in the form of strips of
fdter paper.
NOTE. - On modified Czapek-Dox agu containing saccharose the fungal strains No. 1,2,3 and 4
can be cultured. Modified Czapek-Dox agar containing cellulose can be used for culturing the
strains No. 2, 4 and 5 given below (see clause 2.3.2).
Mineral-salts agar. This is identical with the composition of modified Czapek-Dox
2.3.1.3
agar (2.3.1.1) but without the saccharose.
2.3.1.4 Aqueous mineral-salts solution. This is identical with the composition of modified
Czapek-Dox agar (2.3.1 .l), but without the saccharose and agar.
NOTE. - For the preparation of nutritive media, see the Annex.
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ISO/R 846-1968 (I
2.3.2 Test Fungi. The inoculation of specimens is camed out by applying a mixture of aqueous
spore suspensions of the following strains of fungi :
(1) Aspergillus niger v. Tieghem
(2) Penicillium jùniculosum Thom
(3) Paecilomyces varioti Bainier
(4) Trichoderma vinde Pers ex Fr
(5) Chaetomium globosum Kunze
NOTE. - Besides the above-mentioned five strains of fungi, additional species may be used. If additional
species are used, they should be mentioned in the test report. The test fungi used in preparing the
cultures should in ail cases be Weil defined species obtained from officiai biological centres, the addresses
of which can be obtained from National Standards Associations in each country.
2.3.3 fieparation of the spore suspension. To the well sporulated fungal cultures, which have been
maintained at 30 OC for 2 to 4 weeks, add about 5 to 7 mi of sterilized distilled water con-
taining 0.01 of a suitable wetting agent.* The surface is then gently rubbed with the
inoculation needle for the purpose of transferring the spores into the aqueous phase. By
gently shaking the culture tube, the spores are liberated from the fruiting bodies. This is
repeated with each culture. The shaken spore suspension of each culture is filtered through a
thin layer of sterile cotton or glass wool in order to remove mycelial fragments and other
debris, into the same sterile flask (see Note below). The filtered mixed spore suspension is
centrifuged aseptically and the supernatant liquid is discarded. The residue is re-suspended
in about 50 ml of sterile water and centrifuged. This procedure is repeated three times. The
finalwashed residue is suspended in 100ml of sterile aqueous mineral-salts solution (2.3.1.4).
NOTE. - It may be helpful to work with a known number of spores by counting and diluting, for
example, by separately fdtering each spore suspension, counting with the help of a counting chamber of a
nephelometer and diluting with sterilized distilled water to a concentration of 1 to 2 muon spores per
millilitre. After mixing, the spore suspensions are cleaned as described above.
2.4 Inoculation of specimens
Sufficient mineral-salts agar (2.3.1.3) is poured into suitable sterile Petri dishes to provide a
4 mm depth. After the agar has solidified, the test specimens are
solidified agar layer from 3 to
placed on the surface of the agar (see Note below). The surface, including the surface of the test
specimens, is inoculated by spraying (if preferred) with the spore suspension obtained according
to clause 2.3.3 and the Petri dishes are then covered and put in an incubator.
to avoid any possible dispersal of spores in the air, the test specimens are sprayed in
However,
suitable boxes. When the spore suspension is sprayed from a flask or a sprayer, it is necessary to
shake it from time to time in order to keep the spores in proper suspension.
The spore suspension should be used for inoculation of test specimens with 8 hours after its
preparation.
For a control determination of spore growth, two Petri dishes with the complete nutritive
medium (modified Czapek-Dox agar (2.3.1 .l) or (2.3.1.2) depending on the species) are sprayed
by the same spore suspension and cultured together with the specimens. If during 3 to 4 days no
spore growth in the control Petri dishes is observed, the test is repeated with new specimens and
a fresh suspension.
NOTE. - Covered Petri dishes containing mineral-salts agar are considered to maintain the desired humidity
for 4 weeks. Covers on large dishes may be sealed with masking tape.
* N-methyltauride, sodium lauryl sulphonate or polyglycol ether.
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ISO/R 846-1968 (I
2.5 Duration of the test
The duration of the test is initially fmed at 28 days. In special cases the test c
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