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JIS Z 2801 (2010) (English): Antibacterial

products -- Test for antibacterial activity and
efficacy

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JAPANESE
INDUSTRIAL

STANDARD
Translated and Published by
Japanese Standards Association

JIS Z 2801 :

2010

(SIAA/JSA)

Antibacterial products - Test for

antibacterial activity and efficacy

les 07.100.10;11.100;83.080.01
Reference number: JIS Z 2801 : 2010 (E)
PROTECTED BY COPYIUGHT

13 S

Z 2801 : 2010

Date of Establishment:
Date of Rfwision:

2000-12-20

2010-12-20

DaLe of Public Notice in Official Gazette:

Investigated by:

~Japanese

2010-12-20

Tndu8trial Standards Committee

Standards Hoard
'rpchnieal Committee on

JIS Z 2801 : 2010, .First

Consum(~r

Lifo Products

edition published in 2011-12

Translated and published by:

Standards Association
4-1-24, Akasaka, Minato-ku, Tokyo, 107-8440 JAPAN
In the event of any doubts arising as to the contents,
the original JIS is to be the final authority.
JSA 2011

All

reserved. Unless othel'wiSl? specified, no part of thjs publication may be reproduced utilized
indudin~ photocopying and microfilm, \vithout

in any form or by any means, ekctron1C or mechanical.

permission in lNfiting from the publisher.

N1J/I1N

Printed ill

PROTECTED BY COPYRIGHT

Z 2801 : 2010

rn trod LlttiOf}

........................... , ................................................................. 1

....................................... "1

Normative

:3

TenllS and defi nitions ........... " .............. " ....... , ................... ,., .............. 2

"ted"L)".':";'':>'" ...... , ,.

Antibacterial

,,1

..... , ........................................................ 2

G
5.1
5.2
5.3

Inptllocl ., .. , ............................................................................. ~3
Bacteria to be used
tesL ....................... " ......................................... g
matel'ials, instruments and
......... , ....................... :3

5,/1

Cul.Lure medium, etc ................................................................. 5

5,;'5
5.G
5.7
5.8

'rest operatioIl .......................................................................... 8

of
of
bacteria .. .. .. .. .... ............................ "12
Test resl.llts ................................................................................... 12

sterilization,, 5
Preservation

Record of test

Annox

~JA

bacteda .. .. .. .. .... .... .. .. .. ...... .. .. .. 7

......................................... la

COlnparison
between ~JIS and corresponding
International Standard ......................................... --1;j

(i)
PROTECT.E:D BY COPYHIGHT

Z 28tH: 2010

translation
been made
on
original
Industrial Standard revised by
Eeonomy, 'rrade and Industry
through deliberations nt the ,JaparlBse Industrial
Committee
as the fE'SU 1t. of [1r'oposal
revision
,Japanese rnd ustrial Standard
submitted
of Industrial technology
Antimicrobial Articles
(SlAA)/~Japanese Standards Association (JSA) with the
attached, based on the provision
Article 12
1 of the Industrial
Standardization
applicable to the caso
revision by
provision
of Article 14.

ror

or

Consequently JIS Z 2801 : 200G is replaced with

Standard.

This JIS dOeUIl18nt is protected by thEl Copyright Law.

Attention is drawn to the

may
with a patent right,

this Standard

opening to
the public or
model right
technical properties.
nt Minister and the ~Japanese Industrial Standards CommitLeA lIrA
not msponsihle Cor i(h:~ntifying
patent right, application for a patent

after opening to the public or

technieal properLil~s.

model right which have the

(ii)
PHOTECTED BY COPYIUGHT

~JAPAN

: 2010

INDUSTIUAL STANDARD

Antibacterial products - Test

antibacterial activity and efficacy
Introduction
has be:rm Pl'Pf>af(~rl

This (japanese InduRtl'ial

on th(~

f~dition

of

ISO 221D6 published in :l007 wilh 80nw rnodirications of tlw tpchnieal contents to meet
tcehllology
situation, etc. in .Japan.
The
tents of
with the

<1.nl</.lJu..n,..,

is

or dOlted underlines are the InaUers in which the conA list of modifications
have

in Annex

~JA.

p"~'./"'.LJ,""q"

the test for antibacterial

and
to baeLEH'ia on
intermediate products) of plastic
produets
.....~;~:O.:-~: ,J;,~~.,,~ ...... ";;:;;-.1i;;.;~ :.;:O.;.~.v.,~~;~~~-~ . :.';;'_;;--_:::.:-~!'.:-;~ .x~.~';::, E~xc1ud] n g til e texti.le
and the
J;.

Secondary effocts
tion and

resistance, odour prcvcn'

NQTr;_l._._,~:nLLhe__ PtPd:WJ;1:.::;__!?Q_dg.tRnnined_th!H(.the__ teRt_mf.)Lb..r~ct_fox __t~xti.l~_m:nd!-J.(~.t).

$.hQ~!J.d.bf.;1P.I~JjJ;'_d_fn!m.. Lb.9..iJ:.~J:,~gKQ"..;;1H:1Rf.,..~J.~}~l_d.(l.9.!.3'\;1J.Q..(~~~J.gD.t~tnt[x~;
LQf2j2.QI.JlS..L.19.Q2.Jn9.y..b~.aj)11hf2fJ...

NOTE 2

rrhe International Standard corresponding to this Standard and the

symbol of
of corr8spond(~nce aTe
follows.
2219G : 2007 PlasheS'
s'ul1/u:C8 (lvl0D)

l~1"easuroment

ol itntibf}ctol'illl

on

u"-,.".",-,,,,

In addition.
which denote the
of eorrcspoJ1ckncc in the
eontents betv,,reen the relev~Jnt International Standards and tJIS (up TDT
(identical), J\10D (modified), and NEQ (not equivalent) according to
ISO/lEe Guide 21~1.

Normative references

'rhe following standards contain provisions which, through

sLitule provisions of this St.andard. The H108t. feeent editions
aJnendnl(~nts) indicated below shall be applipd.
rules' IhI' cheJlJicfll
~ns

K 08f)0

SieriJjzed p/;]stir'

disbes

PHOTECTED BY COPYHIGllT

or

in this
eonthe sLandnrds (inelud-

Z 2801 : 2010

tHSK ODiO

Piston

oper;:i (prJ Inicnrvolumetric

cabinets

,ns K 8101

Etlwnol ('[H}.5) (Reagent)

JIS K 8150

(RO/lgCllt)

K 8180

K 82G;3

[u:-jd (RC';1f.{rwtJ
-"/1g.'ll'

\.L,,'"'u ....." ChU

,JIS K 857() Sodium

(Rellgent)

,ns K 9007

PotrJ8,'iillfll

~HS

lJi/.)O{!:lS8iull7

K 9017

,HS H :3505

Volumetric
lor detDrIniJ};Jtioll o1'pII 01'HQllDou8 solutions

(JIS Z 8802

OlcHgent)

Terms and definitions

For the purposps of this Standard, the

3.1

antibactm'ial

3.2

antibacterial agent

agent that inhibits the growth of bacteria

rectIy or as a com pounded agent

a.3

.g.Qt.jJ!.fl~.tm:!aJ..tnml}1lJmJ

~J.5

antibactexia] activity

value whieh shows the

between
antibacterial
by ineubation of bacteria
3.6

g terms and definitions

OIl

the

of products by

it di-

in logarithmic
of number of viable bacteria
and the untreated produet after inoculation f()lJowed

antibacterial effoctiveness

f~ffectiveness

antibacterial product determined from the

The antibacterial product is dptennined that it has the antiba.cterial effectiveness,

when the antibacterial
thereof obtained according to the
method of this

PHOTECTED BY COPYRiGHT

Z 2801 : 2010
Standard is 2.0 or more.
Tn addition, the
of over 2.0 may
applieahle to
terial
upon
betwpen the parties

dptf'rmination of antibacwith dt-divery.

conceI'n(:~d

rnethod

5.1
Tho
a)

test

to be used

of bacteria La
bacteria.

and

tho test shall be as

Lesl

be

StDpbylocoCC1l8 a UiYW8
colj

of bad(~l'ial strain to bH us(~d for the test are shown in ta bin 1. If the bact,erial strain is contrihuted
the age-ney of culture eoHeetion other than that shown in
table 1, it
he
from member
of Wodd Federation for Culture
Collpetions (\FCC) or
Society for Cu1r.ure CoHections (tTSeC), t,md it shaJl
the
bacteria.! strain the same Hories as
shown in table 1.
VV"ULL,H;'"

rrabl(~

Type of bactE~f'ia

StapilylococcllS

Preservation numbet' of' bactm'ial

strain
ATCC H5aRp
FDA 20HP
NBRC 12732

aUr(!U8

test

strain used

or culture collect,lon
American
Culture Colleetiol1
Food and Drug Administration
BioresOtU'ce Information
Departrnent of Bintecl1llo1CIgy of National Institute of Technology and
fi~valuation

CIP G;115n
DSlVI :H6

Collection des Bacteries dE!l'lnstituLPasteur Deut.sche

Sammlung von
und Zellkulturen

Gmbh
NCIB 8625
B~':ic11erichl~l

coli

ATCC 87:jH

NBRC :3H72

elP 53,126

or

Nat,jonaj Collection Industrial and J\13 rifle BncLeria

Ltd.
American 'l'y1Je Culture Collection
Bioresource J 11 ['ormation
Departmen t of Hio'
,-,,"'Y)n,C,II'lCf\,' of National Tnsl-.itute nfTechnology and
Evaluation

DSM 1576

Col1edion des Bacteries dpl'JnsLituL Pasteur DeuLsdle

Sammlung von Mikroorganismen und hellkuJturen

NCTB 8545

National Collection

Gmbh

or Industrial (,j,nd l\:farine Bacteria

TJd.

Chemicals. materials, instruments and apparatuses

rrhe chemicals, matt~riaLs, instrmnenLs and apparat.uses used in this Standanj shall
be as follows, unless otherwise designated.

PHOTECTED BY COPYHIGHT

/1
Z 2R01 : 2010

pxtrad

Purified waLeI'

Yeast extract

Glucose

peptone

E9.1:.mjs,;r.\tRi!:JIJrRt

Lecithin

fQLmj~n:~hinLtf~t

Nonionie surfactant

Polyoxyethylnne sorbit3n monoolentc

r.Q.tnf1tilhl.r)).. {!jb.)~dr.~lgtmf}b.Q.ijrth?1te.JKH~"eQ:J.
Q_~lJ;u:anl&('_d_f.t~.i;:tg(m t_-,?J).~.(;jJ!~~J.J.n.JIS._K .~QQl

niP.D.t!.Hi;?j~H.ll.hysJxngfm.p.hmmh.~1.tQ.H~.~HP'Q.1!.

SD_(h~UI1.Jlyd[Qxi(k__(Nn5JU2
HyslrS!Ghl()xj.c;_nri(t(H(~n

nMki...c:nttm}-,__ nx ..~lUS;!1n.0.. 8t.9P.n~r.>..In~tfJl.~'j.t<.mm1r.t ..mm:.t{~D.

~ tnIJ.n~ f,,_ .eJG
Ow

Plnl.inum Joop

\Vit.h a loop of about 4 mm at the point

Dry-heat

Capable of "'-"I.'V''''''

Autoclave

Capable of
to lOa kPa in ,".

111 oter

temporature from 160C to 180 C

at121 C in temperatun~ (corresponding

,,.."OL:11... ' )

.CfmJ~1rDliDg.tQ.J1S_.Z.88Q.g

Chemical balance

JJJlY!ngJ1~rfQnnaD.{~_~~f.r!1-fQXIlJjngwtQ.JIS..KwQQ5.o__QXwBJlIA~Y!J:.

Clean bench

For microbial tnst

l'vleasuring

PHOTECTED BY COPYRIUHT

[}

Z 2801 : 2010

Incubator

Capable

dish

of

111 m

cham etcr,

~1r

{:.9X\ff!~:mjxlKt9..N9.~.~zQj\.!lJ:.Nn:.}).QJL?IJ.EJs.;jJjt:~!.iu.J.lS.J.\..Q.!).QQ

Film

5.3 Method of sterilization

test tube and
to be Ufwd shall lw washf':d
alkaline or neutral
sufficiently wIth water, then
with dry-heat, or sterilized with higlrpresstll"o steam. 'rhe method
shall he in aeeordanee with the following a) or b). The case WhHI"H the
loop and the test
are stf>.rilizt~d \\7ith
shall he in ac(:ordanc~~ wilh
tho following c).
I'H.Jlld. .L",UJ,U

())

sterilization

to be sterilized shall be placed in a dry-heat steri-

lizer fQ.t:_mLnlin.oX.J11J!n~~.ln.lh~2.!~a~m.Qr.thB.j~eUU)JjXf1tun~.. Qf.17.Q.~.C.QJ:.12.Q.Jn~Jl.Qr..m.m:0.

b) High pressure steam sterilization "Vater is

in an autoclav'~, and
to
which aro in a m(~ta] net basket
metal net shelf in
the autoclave. The lid
autoclave is tightened, 11'"<U.. , .. " ,
121C (correspnnrli ng to 10:3 kPA i n pf(~SSU 1'8) .~'O! '_'''_.''!':_.'_'-_''':._'''':' .. !''':...._'_''_',,_C ... "_'.:::C~::-i?ifi_!-J~~DJ)lu.1mLfQUQw~d..Q.Y:.D.~.tl.lXglb~.!xmU.I.l.Lt_Q.. lQQ..~(~.Qr..gndm;.l..UW.f~xh~l.m~r..y.rtly!d..i&
___

rl .... E19.m~ ..fjJ!?r.ui?:at1nn.... Th~.D.b.L~(:lh.Qr..P.f1.r:t::;.In..b.B..BJm:!J.i.~.B.9..B.hnJ1 ..b.~..nlf~r~f1f1 ..i.njD.. n.gm?

m~.rllf}nhnLnnJ).1fl,-J:~m'__~lJ)l()J.jJ)J.!m_J!!!m~_jJc'3.J:ihtllL.b..\.\:.rt'Jtb.e;It.~d_~Cj)J.fnGjD.ntJ'y',-_mJ_d_fQx.JJ

t~~Lt.lJ.h!t,-jt_fl.h!111.hfl.tJ}.1:w.tH"2d.)yjth__th~.HnnJ.e.(!r._2.B..(~r..a.t'...

Culture medium, etc.

The culture medium, ete.
which the composition is as f()llows shall be usprl. A cornnwrcially availabln artidn may ho usod, if it is of thf~ same composition.

a) Nutrient broth [11500 nutrient broth (11500 NB)] Prepared so that 8.0 g
beef
10.0 g of peptone and n.O g of
urn chloride which at'e weighed out hy
the chpmical balance shall be addpd to 1 000 1111 of
water or ion-exchanged

PHOTEC'l'ED BY COPYIHUU'1'

Z 2ROl : 2010

completely, and diluter! ;")00 times with purified

t.o G.B to 7.2 (2fj
with a SOrlilHl1 hydrox
the pIT Illcter, and it
be
ide solution 01' a hydrochloric
pressure steam. If it is not used
after preparation,
'-"i'''Y-'''''''''' elL () tcoll)erature ()f 5 0(; to "ll) O(_;.~ 115 ()() NB Lllat llas
Olle
JH'Pllaratiol1 shall not IH~

wa/er, then mixed and

water. 'rhe pH

shall

Nutrient agar Pl'epand so

5.0 g
10.0 g of popton(~
5.0 g
sodium chloride shall be addt~d to 1 000 mI of purified water 01' ion-exehanged wat.er, and mixpd. 'fhe
thm-pof shaH be adjustefl to 7.0 to 7.'2 (25
with a sodiunl
hydeoxidc solution or a hydrochloric
solution . .l.t}.._Q_R_QL!X!yy.df,H: __(;tgaL~hf.tlLht:
adJJ.0.d and dissolv0d by heating, n cotton stopper
be
and it shall be sterilized with high pressure stearn. If it. is not
immediately
preparation, it
shall be preserved at a temperature
5 C to 10C.
nutrient agar that has
been kept for one month or longer after preparation shaH not be used.
e)

Plate count agar

Prepared so that

g of

tryptono and 1.0

g of glucose \vhich are weighed out by the chemical balance

ht! added to 1 non
ml
purified water or iOlrexchangpd watE>:!" and mixed.
pH then~of
he
adi\H~;.tD.fLtQ 7.0 to 7.2
0(;) with a 8odiumhydroxirn~ solutjon or a hydrochloric
acid solution by
thQ pH meter. 15JLg,QLnnwdm::_ng!Ic~'?lw_UJ~fu!dJl(td a nd dissolved by heatlng, and it shall be storihzed with high pr(~ssure steam. Tf itis not

immediately after preparation, it shall be preserved

a temperature
to 10
The plate eount agar
has been kept
one month or longer
prepara tion shaH not he used.

d) Slant culture medium Prepan~d so that f) ml to 10 ml

the nutripnt agar of b)
which has been
iminary wiHmed and dissolved shaH be poured int.o a test
a Gotton stopppl' shall be
and it shall be steriliznd with
prf'SSllre stnam.
stnrilization.
t(~st tube shall
placnd in a
room at ~lant
about
15 to the horizontal plane, and the content shaH
solidifiod. Tf it is not used immedjatelyafter
it shall
preserved at a temperature of' 5 C to 10
If then:~ is no eOl1chmsed \~/aU:~r, the content shaH be dissolved and solidified again,

then used. The slant culture medium Lhat has been kept for onp month or longer
after preparation shaH not he
e)

SCDLP broth Prepared 80 that 17.0 g of cas(-)in peptone, 8.0 g of soybean peptone,
5,0 g of sOflium chloride, 2.5 g
disodium hydrogr.n phosphatE',
g of glucose and
1.0 g of lecithin which are weighed out by the chemical balanee shall be added to 1 000
ml of purified water or ion-exchanged water, then
and dissolved. 'fhen, 7.0 g
nonionic surfactant shaH
added and dissolved, 1'he pIT thereof sha II be adjusi.cd to K8 to 7.2
C) with a sodium hydroxide solution or a hydrochloric: acid
solution by using the pH meter, and it shall he sterilized with high 11I'essure sLearn.
it is not us(~d iInm(~diBtely after preparation, it sha.ll bp presnrvpc1 at. 5 C to
10C. The SCDLP broth that
been kept for one month 01' longer after preparw
bon shall not
used.

f)

Phosphate buffer solution Prepared so that :34.0 g

PROTECTED BY COPYRIGHT

pntassiUJH dihydrogen phos-

Z 2801 : 2010
weighed out by the chemical
shaH be added to 500 ml
pu
th('Il mixed
pH t.hereof
adjusted t.o (j.B to .2 (25C) with a sodium
solution or ()
chloric acid solution by
tho
met~r. Ii'urthel', purified water 01' ion-ox
water
to
it. 1 000 m1, and it
II be
with
high pressuro stt~am.
buffer solution rhat
month or longer

wa tel' or ion-exchanged

g)

5.5

Phosphate-buffered physiological saline The phosphate buffer solution f) (D.Sf) r~r)

chlorid(~ solution) shall t}(~ diluted 800 tin1fI,S with Y,nu.c",'F":'"!/",,.,>",,,~~ salinEL If
""'1'>01.[1 into Lest
necessary; it shall
sLop
pel'S shall
and it shall
sterilized
not
llsed immediately
preparat.ion, it shall be preserved at 5
'rhe
phosphate-buffored physiological
that has lH:~8n
for ono month orlongor
after preparation shall not be used.

Preservation

bacteria

rr10nth or the transfer, the next l.rclIlsrer shaH he carried nut. in a same way to he the
passage eu lture. Tlw number of passages shall he up
times counted from thi;~
original strain obtained
t.he agency of culture
Furthermore, that
for one llHmth or nlOr8 from the last t.ransfer shall not be uspd for the
trans-

PROTECTED BY COPYRIGHT

Z 2ROI : 2010

CondfXlsc:d wator

Slant eulture

l1wclium

Figure 1

bacteria

'rest operation

5.6

Bacteria shall
handled asoptically, and attention
bo
to the eontaminw
personnel, instnunents and V\forking environment with bacteria as ColA
cabinet shall be
if necessary.

I ion of

a)

Pro-incubation of tost bacteria One platinum loop of bacteria

be
from the preserved bacterial strain of 5.5 to the slant culture medium of 5.4 d), and
incubated at a tum perature of
1 C
16 h to 24 h in
incuuator ..Furbacteria shall be transther, from this incubated bactetia, one platinum loop
to a
slant cultu:re medium and intubated at a temperature of 85

b)

pf(~paration

of test piece The preparation of test pipce shall be as follows.

1)

1'h8 flat part

produet shall be cut into a square of 50 mm 2 mm (within 10
mm in thicknpss) and made as the tost pieeo or standard SiZfL If it is diFficult or
impossible Co cut lJl(~ product into this squarp, a t,est piE~ce of
and size other
than specified may
used if it ean be covered
a filln of
surface area of
/100 Inm i to 1 HOO n1mJ.

2)

Untrf)c) led test

shall bE' r~ut ouL from the antibacterial untreated produeL or
the film. Among six untreated test pieces, j,hree
used for the measurement of number of
bacteria immediately after inoeulation with test ino(:ulurn and another three shall be used for the rneasurement of number of viable
bacteria aftet.' the incubation for 24 h.
micro-organisms,
tamination bet.ween products and filth for the preparation
test
Itis desira hIe to sample the LesL
from the lwoduct itself, but. if it is difficult. to

PROTBCTED BY COPVl{lGHT

n
Z 2801 : 2010
pt'nr>ar(~

Uw

pi'(~vared

tE~sl

because

from

raw mat.eria'! and the

the shape of' the

test. pi<:'ces may bp
to a plaLe shapp with the S(I me

n"(""-{),"'''l

{thr:\lE;.ltirl{:('J~)..<;HJ.mnt._~.~:_ln.~.J)J).r.<:::.lL..th~..citm..~.b.ul).JJg..1J.i?~.~i.J~I!:.?f!:

~n1f:: ..w.hnl~ ..~.9xf~w.()..nf..tmit.ntm:t~.n.R) ..t'lH!Jl.tn~_Jjgh.uY_.Y!:~l!~;~t,

c)

dissolution of surfHee coating a,nd ehr

If changes snell (iH Hoftening- of test
tion of components occur after
tl'E~atments and it is considered that. these
tl'eatnlCIlts affect the test
test piece shall
cleaned by another appropriaLc mcthod~ or used (1sit is without e1eaning.
Preparation of test inoculum Oil(~ platinuln loop of
the testbaeteria
pre-incubatcd in
shall be
in a smaH amount of l/fiOO NB of 5.4
a), and the bacteria concentration shall be est.imated with
microscopic observation 01' other appropriate n18thorL rrhis inoculum shall be diluted with 11500
NB <.lppropriatel:y and adjusted so
bacteria eonermtration heeomt~S 2.5 x liP
to 10 x 1l)5 cells/ml, and this shal1
used as the test inoculum. If the test jnoeu
he cooled on iee (0 e) and shall be used
lun1 is Ilot used imnwdiatdy, it
within 2 h after
e)

Inoculation with test inoculum

lows.

inoculation with test inoculum shall he as fol-

1)

Each test.
of c) shall bc"' placed in a sterilized petri
making 1.he test surup.
test. surl'acE'
the
the product on which antibacterial
treatment is performed. Even when the antibacterial treatlnent is
to a
the cross section shaH not be used as the test

2)

Exaetly 0.4 ml of test inoculum of d) shall

taken with a measuring pipptte and
instilled onto each test
in the
dish. Tho volume of inoculated inoculum
on the test piece whose size is other
standard size shatl be proportionaH.y
area
covering filnl. Even if the test
is of
divided by the ratio of
standard
when
volume of inoculum based on the provision is inoculated
on
test pioee of vpry good wE~U.a bili ty sueh as
tile,
slant and the inoculum ma:y escape from
edge of
film may move at a
the film. In this case, the volume of inoculated incxulum may b() rednced up to 114
of the
volume. However, even when the volume of inoculated inoculum is
concentration inoculated on the te:-::t piece shan be tt2 x HP

3)

The instilled test inoculum ~hal1 be coven:;d with a film, the film shaH be gently

PHOTECTED BY COPYHIUHT

10

Z 2ROl : 2010
TH>':H-,C'I.,d

so that the tf;st.inoculum

across the film while paying atUmtion

it. doc's not ::;piH over

the edge of filnl, and the Ud

the petri dish
rrhp standard size of
be the square of ,10
plaeefl (se{~
mm 2 mm. If the test
is not ihat of t.hn standard
Lhe size shall be
jllsted so that the film can be placed within 2.5 mm to 5.0 mill from
of
the test
sizp of film shall not UP
to loss th811 400 mm~. Fur
to adherE' the filrn
nee the shape oftl:~stis not
LheY',if it is
nat, if the
inoeulum spreads over the test
without covpring tlH:~ film
sinee the Lest
is hydrophilic or water
or thf~ 1ikf~~ filA proeess of
covering Uw film may be OIniLt.('d. \VhfUl thf~ covering process of film is omitted,
the standard size of test piece which is
in 5.6 b) 1) shall b(:~ th(~ squarE' of
40 m.m 2 mm (within 10 mm in
\,I1.J.'-.""..<.<"",:> ....O/

inoculation with test

if it is difficult to
the leakagl~
the edges of the fihn on
case where the slrd'ace of sample is vpry hydrophilic and th('. like, Ihe volume of inoculum may be reduced up 10 0.1 mL In
Lhis casp, the concentration of bacteria eelh:{ in inm.::ulurn shan be inereased to
provide the; samc: number of baeterial cells as that of inoculated inocuhull of
rnal volume.

Unil: 111m

0.1 m1 of test
inoculum

Test piece
\

Lid of

dish

Petri dish

0.4 ml of t.cst
inoculum
("'~

+!
o

-.::!"

r",

+1

c
on

Test piece ./

Petri dish

Figure 2 Instillation of inoculuIll on test

PROTECTED BY COPYRIGHT

and covering by fUnl

11
Z 2801 : 2010
f)

Ineubation of inoculated test pieee with test inoculum The petri di8h
test
Lhe test inoculum (three
test piecos and
shall bE~
a t.ernperature of :3;,) C 1 C
1. h.
of a product is eval
from the anactivity obtained
the test at
incubation tempera,:,p.,,\.,j.J..C\..u here; however thH test at the tempF~rature established
considering
actual use of
antibacterial product
a room
temperature) may be carried out together if
parti(~s
toneerned with delivery.

g}

Washout of test bacteria inoculated

shall be B H follows.
1)

Test

The wash-out

inoculation with test inoculum For three lmtn:"uted

inoculation with test inoculum, tho coveting film
on another
dish
wit.h caution
so that
inoeultun dops not spill. By adding 10 Inl of SCDLP broth of 5.1 e), the
inoeulum on t.he untreated test piece
be
out with ()
and this wash"out inoculum shall be reeovered completdy. The
proceeded to the measurement of the
number viable bacteria.
immediately

test.imnwdiately
and the tust pieee shan be

.I.LH."","A.L>.L'.l-\

or

2)

rrest pi ece

a)

.~:Px..tbg..ww?JJ.:Q!:J.t.Q[.t~;:;.t.JHJ.\:J~~rjx~,-.Ul~_rngthrHJ.~'?lL<.:b._tJ)).:1L.th5:.S:Qy~!:tnK.nlm..?nsJ.. ~bJ)

incubation

For the test

after the incubation of

the test
bartkH'ia shall bE:
out in a similar way to 1} 'rhe
shall be i mrnw
dia
to Lhe measurement the number of viable baeLeria.

t~:},":it.niH~~ft.f!'(~_I]J_()KB!J._iD_.::I_.;1tBJ:jli_~_~~l_t1.t!~m.(H:.h0X_l}.9.~~dl_h.Y_.l!Bjng)itRr:Hj!l~d_.t}~~~ft!:.\~.r.~

wiLh.mlgJjmt~Q.!1~fl.Lth.0.j.nn\.~I0,h.nu_dQ~~~..nQL!3.P.UL1U.mJ..QX.S(;J)JdJ~_hn~lh_!n ..S..J..~2.j~~

wash out the test bat;Leria with 10 ml of SCDI. P broth because

charaeterislies of the test piece, thf' volunw may

or the size

and

increasod.

h) ~leasurement of number of viable bacteria by agar plate culture method Exactly

1 ml of
washings of g)
be
with a llwasuring pipette ano added in a
test tube containing 9.0 m] of phosphate-buffered
saline of' 5.4
and
sufficiently mixed. Then, 1 nd shall
this test tube
a now meas'
pipette and add in anothf~r test tube containing B.O ml of phosphatwbuffererl
phYbiologien 1 saline, and
t1y mixed. These
sha il he repon ted to
prepare H)-fold serial dilutions. 1. ml each of the washings
be
into two sterilized
dishes.
~c~~~c~~.,J;~,};~.:~~ ,,:;"'!.,~~, ... ""',~ .....,,'!....;::~_ . .,,._"'_._:-,,:'-,, ... ::?'.
tll.9.J)J.<;1.t5J_.~.9_\lnt~ ..c}'.fGJx..QL5.,1..c).. )Yf!Xm.0.<J.. Qt.:J.<3.. ~c._tJ).. 1.8..~.C ..$haU.b~ ..~~d~k~J.. ~1n.d.~!JjIi:.
_"!;_':c...

IJ>'"'~'U'''''

room

the lids, the

djshes
be
the culture medium, the

PHOTECTED BY COPY1UCH:lT

as
arc at a
dishes shall lw

1.2
Z 2ROI : 2010
turned over, and incubated in the jncubatm:' at a ",,",
h to
h. After incubation, the numb(~r of colonies in a

,Lq_"_, "."

vv'hich :30 Co

of 85C
dilu

1 0(;
40
petri dish in

number of eolo-

colonies appeal" sha11

nies "is
than :30 in the agal' plate '-'''--'fJ'_"J"'>C''v"'-'
number of
shall be
If there arc not any
[urInations in any agar
then t1< It!
hf~ r(~eorde(L
, if thc; numbHf
of colonies is not inversely propod.ional to the dilution ratio, since it is considered
1,ha t
formation of colonies is inhibited by tho
of the antjbacteriaJ a.gent,
the number
shall hn determinerl using a mt~tho(l w h1Ch
colonies
affected by
antibacterial
with
use of an inaetivating agent 01' dilutjon.

WUJ.~.Q!:.ChfJl).t~1!:_2JJfJ~~tQxj_~_~!_SnQjJ_0.gr~.inJJ.iQ~ttm~_b_~G.tQr.t~ll ..L_J~fJJ~Ql_1~{t.<;___
L()xi_{JI.J;Q9xlt;..nf..Sh{tng_Q.nJ.. JY1Qt~hQ.dEl._Df_AngJ.Y.$j..iu_J:~md .. SnXtlty_J~~'gul.g_-_

l.j.\m .. (~J).~H) .. H~J).<:Jx:y5.to.GJ.l. ..b.Y. ..t.h~... f).ny.!ntnDI~.nu:.J..l .. Un!JJ.th.J3~1.r:~fJY...~~f..1JJ.t~

M_i.nj;')try'.Q.f_HH~J.th.JAnd._\!:~Jf{lX~-,-.J.fm5!n~

5.7

Calculation of ,u...... J..'--'-']'-~ of viable

..

The numb(~r
viable
cording to equation (1).

III

uU',,''-".LLU

('" x 1) X pI"
A
.\r

n'~'"'l""''''''

shall he obtained

counts of colonies measured ae-

.......................... , , .... , ... , ................. ( 1)

V:

bacteria (per 1 cm 2 of test piece)

number of
count or c:olonies (average count colonies of two petti
dishes adopted)
dilution factor (that of dilution dispensed into
dishes adoptee!)
volume SCDLP broth
for wash-out (m])

A:

surface ~uea of eovering fjJm (~Jn~)

111

C:
D:

}}}.H1~!.~:~:tf&.Yih~}n~_t.h~~.~~Q.y:~:xj_ng.nlm. .tB..Qmjtt,g_~1_.ln..9._ fi_~)..?)~_A_~hJJ.n.h~.}.th!Y.~~~n:f~.(:.Q..~.nLt;.\

r()_\!D_djJ1g..Qff_.thf_.thjKd __ ~igDjJj~f1n_t.Jjg\lx.E;'-,__ Wh_t;n ..th.~._~~mnt._QLrc9J.Qnj_~J?__ f?5_$__ ::S:.r~, ..r/_.i.:;"

L~l.k9.n.EJ.))..f~.l~~l ..nnd.th.f2.J}~!mJ)J:X_D.L)!_iB_hlf!JH)g.t9.f.i.~l_fihJ.IJ.IJ).t;;..(:DJ.f.~llfl.tttd.f;nr.r.~~~f}f}D.d!D,g,.!g.. E.

5.8

rrest resuJts

'rhe lest results shall be as follows.

a) Detorm.ination of conditions of test validation Whpn
fol]owing threo tpst conditions are all satisfied, the test shall be det.ermined to be valid. Unless an Lhe con-

PIHYl'EC'rED BY COPYltlGHT

l:~

Z 2801 : 2010
Ih8 test shaH be determined t.o be not va lid, and. :l re/pst Rh:dl

ditions are
be carried out.
1)

for

logarithmic valuE;

trw

nllmhel~

test

of viable
-==------'-""'"- ~

where,

0.2 .................................................................. (2)

maximum logarithm number

viable bacteria

minhnum logarithm number

average of
lest

b)

numbers of'viable

2)

The av(~rage
lmtl'eated test

a)

The nunlbf'I' of viabln .... '".,'''''-~''' on untreaLed tps!. pieee

than 62 eells/em2 for all
test

of three

number of viable baet.eria immediately af!.er inoeulaLion on 1,h('

shall
within the range of 6.2 x 10:0, to 2.5 x 104 cells/em 2

Calculation of antibacterial activity \\'h8n the test has been determined to

valid, the antibacterial activity shall lH~ obtained according to equation (8). The
value shall
to the first decimal place by rounding the second decimal
placE' down.
the number of viable bacteria 18 11 U:;3l!, it shall
taken as
HO.6~5tt and the average of logarithm numbers shall bp ea1culated.
)

R
where,

(At -

)= U

At" .................................... ,. .. <:0

antibacterial activity

average of logarithm numbers of viable baeteria immediately

inoculation on untreated tost
Ul

At:

()

average of logarit.hm num bel'S of viable bacteria after inocuiation on untreated test pioces aftel' 2;1 h
average of logarithm numbers of viable bacteria nL'Ler inoculation on antibacterial test picce after 24 h

Record of test results

The following matters shall he listed in the test results of antibacterial produeLs such
as plastic products.
Number or ti

of this Standnrd

b)

Commencement date of test

c)

1'ypC' ,
test pjcce

Type,

shape and thickness of antibacterial-treated test piece and untroated

shape and thiGkness of film

PHOTECTED BY COPYIUUHT

Id
Z 2R01 : 2010

e)

of test bacteria

f)

pf(~servation

g)

Volume

h)

number of baete:rial strain

test inocuhnn
bacteria in test inocuhun

of

i)

Cleaning method

.1)

Hcspeetive
laboratory, nanw

k)

Any

ch~viation

antibacterial
and date of test

5.8 b), identification

rc~pol't

this specification

Bibliography
~TIS

1~)()2

Testing lor fllliiiNJcler.i8l11ctivit:f,' and el'fieacy 011 textile products'

PROTECTED BY COPYHIGHT

JA \,l.J...l.LV~~J..laL-J.
narls:on table between JIB and corresponding
JIS Z 2801 : 2010 Alltihacteli;;zi products -

Test [OI ant.ibactclial activit)' Elnd (Ii-

m Requixcrl1ont:s in .JIS

No. and
title of
e1anse

Content

1 Scope

Testing method
antibacterial activity
and
to bacte'
ria on the smface of
antibacterial products

~
~

t::2

(IT) International
Standard
number

(UI) Roquil'cmcn ts in
1 nte-rna tiemal Standa rd

No. of
ehtuse

t:

b:
t-<

ate
arC'
speeified excluding the
textile produets and
the photocatalyst
products,

C
'"e

....::
~

;...

S
,.....
~

i3 Terms
and definitions

ISO 22196 : 2007 Plastics - .1lIeasUl'0111cllt O[;l11 tiba cteri!:li !:lcti,ity 011
pit:.lstic:,; s'urf'tlces

C./1(~T'

"'C:

UVCUU,<.CI..L

Terms and definitions

used in this Standard

are specified.

:3

Content

(IV) Classification <Iud details of teehnicnI

deviation lwtwecn JIS and the Intern.ational Standard by clause
Classi.fie~r

(V) ,Justific8tion fo!'

technical deviation and
future measures

Detail of technical deviation

bon by

clau:::!E:'
Evaluating method I Addition
of only the hactcl'ial
activity of antibac"
terial-treated pla::;tie
products \.lIn,'UUliH::;
intermediate
nets) specified.
HO\:\18Ve1'. it is mel!'
tioned in "NOTE'!
that it may also be
suitable for other
non-porou.s materials.

There is no substatr
tial deviation from

JIS,

I Addition

In ISO Standard, the object

product is limited to only
plastics products. Also in
ISO Standard, there i8 no
criteria of antibacte.rial of-

The limitation of object

product in ISO Standard
is based on such system of
ISO
as being
ilw8stigated in the committee
In Japan, it demanded
from the consumer that
the criteria of antibade"
rial efficacy is 8pecified
"vhen antibaetC'l'ial products al~e evaluah~d.
There no substantial
deviation.

The definitions of antibacterial treatment and antihaG"

terial produd. are not described. in ISO Standard but
only in JIS.

It hased on the technical

evaluasituations in
tion of antibacterial activo
ity in Japan. There is no
problem because
definition of "antibacterial" is
the same.

N
L~

cr.:
o
l-"::

IS
t--,:'
::J:J

en H.equirements in JIS

(II) Inter

national
Standard
No, and

tivenesI:J
'"t:

(Iv) Classification and details of technical

(v) Justification for the

deviation between .TIS and the International Standard by clause

technical deviation (md

future measures

No, of
ela usc

Content

Concrete numl~rical
valUE> is specified as
tbe criteria of antibacterial effectivenC85,

8,4

The antibadcl'lalactivity values used

to define the effectiveness shall be
agreed upon the
parties concerned
with delivery.

Addition

;).2 Chomicals. materials. specifications of

instruments. apparatuses, grades. etc. are
specified.

4.2

It is approximately
same as ,JIS, however grades are not
specified in ISO
Standard.

I Addition

0.3 a) Dry-heat sterili-

G.l

Content

title of
clause
4 Antibacterial efree-

numher-

OIl) Heqllirements in
International Standard

Cl ass! fica -

dause

li
q
f-j

tr::

t;;
b:
~

C
""C

>-<
~

C
::c

5 Testing
method

Detail of technical deviation

bon by

There is no substanLia] devia tion from

,JIS,

For the chemicals, matl!l'i"

als, instruments on which

JIS is existing respectively.

the corresponding JIS is
referred. For the purified
water. the ,Japanese Pharmacopoeia is referred, In
ISO Standard. the compositions of heef extract. and
peptone arc specified in
Annex A.: hov.revcl' in JIS,
these an! speclfiNI to be
microbial test use.

zation lnetbod is specified.

In JI8, the clIltibacterial

I It is Hot specified in ISO
activity shaJJbe 2.0 or more. Standard beeause there is
the possibility that the
criteria is altered dependlng on individual conntry
or product.
There is no substantial
clevia tiem.

I Addition

There is no provisory sentence .in ISO Standeli'd.

It is b:u.;ed on the technical

situations in the evaluation of antibacterial activity in ,Japan. and there is
110 substantial deviation.

It is based on the technical

~ituationsin the evalwltlon of antibacterial activ.ity in ,Japan, :1nd there is
no substantial deviation,

1--'
t~

':::?

1--'

~,

c.

Requirements in JIS

No. and

Content

national
Standarcl
number

(III) n.equjrements in

(IV) Classification and details

(v) Justification f(w the

International Standard

rl,:",,,,.T1(),"r1

tel:~tl1tll(.:8J

future
No. of

Content

6.2

There is no Hubstantial devi~ltion

title of
clclUS(:,

method
(continued)

"'C

;::c
C

r-::

t=:

r::
;-::

5.;3 b) High prCl:fsure

steam sterilization
rnetl10d 18 specified

technical
between JIS and the Intel'11a-

Classifica'
tion by
clause
i\.ddiLioll

technical deviation

The conerete operation

method after heating

d.escribed in ISO Standard.

JIB.

c) Flame steriliMtion method is specified.

Detail

Addition

The flame Hterilization

method is not described in
ISO Standard.

Alteration

:\ftcr pH is adjusted. agar

added and dissolved.

t:::::
~

tc
r-<
r'"'-

es

"'C

;).4 b) Nutrient
propn 1'8 tion !Jlethod
specified.

4.2.8.:3

Agar is added and

disf.'ohE'd, then pH

deviation and

Inlc~a13Ulres

I L is bUbeu 011 the lC'"!1Jeu.<..cU

situations in the
tion of antibacterial activity in Japan, and there is
no substantial deviation.
I t is based on the technical
situatiol1s
evaluation of antibacterial activ'
ity in ,Japan, and there is
no substantial deviation.
altered. fbI' the pur
of\'Vorkability in1-

adjusted.

e
10-

ment. There iH no influ'

ence on the
operation and. result.

....:

SA c) Plate eount agar

prepmatioll method is
specified.

4.2.8.4

Agar
and
I Alteration
dissolved, then pH is
adjuster!.

After dissolved.
pH is ndj LIsted. t.hen
added.

It is altered. for the l)urof worlmhility improvement of pH adjustno influfi)llt)wing oponce

eratie)11 and n'csult.

~
t:-:;
(f-;

0
l~

......

i-'

-:1

t~\

'l)

'::>

(I) Requirements in JIS

No. and
title of
clause

Content

5
Inethod
(colltin ued)

5.5 Tl'an1:lfer of bade-

(II) IntE;l'"
national
Sta.ndard
number

(III) Hequil'ements in
International Standard
No. of
clause

Content

6.4

PrHserva Lion con(li-

ria and preservation

method thereafter are

ClassiJica'
tion
dauAe
A. ddition

strain and th!;:!

The operation proeedure of

PUb'

sage timing after

tral1Sfl'r are same

~ .pl:.~\",L.Ut,;U4

c,...;:

(V) Justification

the

technical deviation and

future measures

Detail of technica 1 devintion

bacteria transfer is not de'

scribed in ISO Standard.

Lion of original

:t'; ...1

"""'
~

(IV) Classification and details of technical

deviation between JIS and the Interna'
tional Standard by clause

is hased on the technical

situations in the evalua'
tion of antibacterial activo
ity in ,Japan, and the.n.:
110 substantial deviation.

those of JIB.

t:rE

t;:;

5.6 b)
method of test piece is

7.2

There is no substa nt.ia1 deviation from

/\ddition

The alternative method

when untreated tetlt pieces
cannot be
is not
described in ISO Standa.rd.

based on the tech niea1

situations in the evaluation of antibacterial activity in ~Japan, and there is
no substantial deviation.

Alteration

"\Viping
ethanol is
1jupposcd in JIS: howcvel' if
it cannot be appJied, dean"
ing is omitted
otn(lr
mothod carried out. In
ISO Standard, no "',LI.."(u,<"U&
is presupposed; ho""ever
wiping with ethanol,
may be applied,

It is based on the technical

In JIS. thE! conventional

method llsing a stollUlcher is
also described in g)

It is hased
tht, Ipchnieal
sit.uations in the evalua'
tion of antibad:erial activo
ity in ,Japan, and there
suh::;tantial deviation.

JIB.

'"'"-

"'C

K:

:::c:

C
iJ:
~

:3.6 c) Cleaning method

of test piece

7.2

There is no substantial deviation from

JIB.

5.6 g) Wash-out of
bacteria inoculated is
spocifiod.

7.G

BubstanThere is
Linl deviation from

JIS.

Addition

situations in the evalu:r

titm of antibacterial lU:tiV'
ity in ,Japan. and there is

no substantial deviation.

r-'

t~

1::::
I-'

,-.,

'l)

(II) Inter
national
Standard
numbet'

(I) RequirementR in JIS

No. and
title of
clause
G Testing
method
(concluded)

"i::
~

Content

(IV) Classification
details of technical
dtwiation between JIS and the International Standard by clause

Requirements in
International Standard

of

Clussifk1:1 .

Content

.7

{'i.S h) Measurement of
number of viable bac'
teria by agar plate
culture method
Provision:
methocl of pIa to count
agar

clause
There no substan- I Adchtion
tinl deviation from
~JIS.

In JI8, the wHl'lning condispecified. The range

of amount of plate count
agar is also extended.

5.7 Calculation of
mUD bel' of viable bac

There lS no substantial deviation from

teria

JIS.

8.1

Addition

An

in which, when
rnunber of coloniel:> i.s
"<1", the number of
bacteria is indicated as
"<0.G3", is descrihed.

It is to indicate the HumLee of viable bacteria in

the unit of cm~l for making
to be more clear, and there
is no imbstantial clevieltion.

Addition

Whl:-m the film is m..;ed for

the untreated
piece, the
condition of the number
viable bacteria
2,1 h is
additionally mentioned.

It is based on the technical

situations in the evaluation of antibacterial flctiv'
it.y in
and thert~ is
substantial deviation.

c:
~

>-<
:;;

8' .""q

5.8 a) Determination
method of conditions of
te8t validation is speciGed.

C
:::c
J....:.i

Overall

1'he1'8 is no 8ubstalr
tial deviation from

JIS.

of correspondence hetween eHS and International Standard (ISO 22196 : 20(7):

NOTE 1

based on the technical

;;;itua.t.ions
evaluation of antibacterial activjty in Japan, and there is

Bubsta.n tinl devia bon.

t;2
v

Detail of technical deviation

chwse

l-::

ts

(V) Justification
the
technical deviation and
future measures

Eub'columns of classification
Addition: Adds the specification item(s)

clause in the

table indicate

MOD
follo'vvs:
~

content\s) which are not induded in International Standard.

tv
(f,

.- Alteration Alters the specification content(s) which are included in International Standard.
NOTE 2

Symbol in. column

overalJ

of correspondence between ,HS

International Standard in the abO\'e

indicates as follov{/3:

l~

--

MOD:

international Stand:lI'd.

r-,"''-'"
.............

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