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êRENCH STANDARDNF T 72-150~ lrp

Water miscible and neutralizable antiseptics and disinfectantsused in liquid form

Determination of bactericidal activity

Oi lution - neutr~ I i sation methodF: Antiseptiques et désinfectants utilisés d l'état liquide.

miscibles à ¡'eau et neutralisables

Détermination de i 'activité bactéricide

Méthode par di lution - neutralisation

French standard certified by decision of the Director Generalof AFNOR on 5 October 1987 to take effect on 5 November 1987.

Supersedes the cert i f i ed standard of the same number ofMarch 1981.

CrirrespOndence: At the date of publication of this document. there is nointernational stand~rd aealing with this subject.

Analysis:

Oescriptors:

This standard makes it Possible to give an opinion on andcompare the qual i ty of products used as antiseptics anddisinfectants. The activity researCh is carried out in alaboratory (in vitro) under specified conditions and makesit possible by thp dilution-neutrglisation method toassess ù reduction of at least 10 times in the number ofliving cells belonging to selected strains of bacteria.

Intern~tional Technical Thesaurus : Disinfectants,

~ater miscible products. bactericides. effectivness.

laboratory tests.

Modi f ications:

Correct ions:

Compared with the prpv i ous ed it ion. i mprovement of the pro-cedure. introduction of two test Possibilities using either 5strains (spectrum 5) or 4 strùins (spectrum 4). and alterationof the expression of the results.

P"blu.hcd .a~ dULl'lw&.d. by l'AUOCLatlOft "'uac.aLM de Nonna.LlOft tArNOR).

ToW' ..:u'o..... t:l!d.. i. 9'%010 Pa. l. O.f.nM. T..i: (1) 42 91 )) ~~

TtinMAllOlI: T.C'balcal H~ID 10 Earwlrtu. S",nir. nl asi

11) AFNOR 1988

313

Water miscible and neutral izable antisepticsand disinfectants used in 1 iquid form

Determination of bactericidal activity

Oilution - neutralisation method

NF r 72-150

~1C£7

FOREWORD

This standard describes a general method intended for:

- Finding out whether a I iquid product proposed as an antiseptic ordisinfectant has or does not have a spectrum 4 or 5 bacteriCidal

~ctivity under the laboratory conditions defined by the standard;

- Comparing products with each other under reproducibl~ conditions.

The attention of users of this standard is drawn to the fact that the

practical conditions of use of the product to OP pxamined are always

different from the laboratory conditions defined by the standard, with

. regard to:

- the nature of the medium in which the product must act,

- the nature and quantity of the micro-organisms.

- the temperature.

- the nature of the interfering substances.

- the rate of destruction required for the micro-organisms.

and it is essential for practi£al applications to complement this

standard by other tests based on standards NF r 72-170. III and/or

NF T 72-190 cr Gther standards Deing prpparpo.

- 2 -

CONTENTS

2

3

SCOPE

FIELD OF APPLICATION

REFERENCES

4 DEFINI T IONS

4.1 Bactericide

4.2 Determi nation of bactericidal ~ctivlty

5 PR I NC I PLE

5.1 Pretes t

5.2 Actual test

5.3 Performance of the tests

6 STRAINS

6.1 Reference strains

6.2 Verification and preservation of the strains

6.3 Culture of the strains

7 WATER AND CUL TURE MED I A

7.1 Water

7.2 Culture media

7.3 Bacterial suspension preparation liquids

7.4 Neutral izing agent

3

315

'IF T 72-150

Paqe

5

)

6

7

7

7

8

'3

8

8

9

9

9

9

10

::.

10

1 I

12

,'IF T 72-150

8 APPARATUS AND GLASSWARE 128.1 Apparatus :28.2 Glassware and small items of equipment 128.3 Steri I ization i 39 PRETEST l39.1 Preparation of the bacterial suspensions 139.2 Counting of bacteria in t~e suspension 149.3 Testing of the different neutral izing agents lS9.4 Incubation 169.5 Read i ng 169.6 ~alidity of the bacterial suspenSion and choice ofneutra liz i ng agent 1710 ACTUAL TF.ST 1710.1 Preparation of the bacterial suspensions 1710.2 Counting of bacteria in the test suspension

10.3 Validation of the neutralizing agent

i 7

18

10.4 Product tests and counting of the surviving bacteria 19lO.5 Incubation 2010.6 Reading 2011 EXPRESS I ON OF RESULTS ¿¡i 2 REPORT 21

APPEND I X A

APPEND I X B

¿J

26

APPEND i X C 29

- 4 .

316

NF T 72-150

1. SCOPE

The purpose of this standard is to describe a method for determininq the

bactericidal activIty of water miscible and neutralizable antiseptics

and disinfectants used in i ¡quid. form, by the dilution - neutralisation

tecnn i que.

2. FIELD OF APPLICATION

It should be borne in mind tnat bactericidal activity is the property of

d product whicn kills bacteria under specified conditions (NF T 72-101).

This definition is limiting: firstly, It refers only to bacteria, in

vegetative form. and not to bacterial snores. or to fungi or to viruses.

Moreover. it states that the conditions of hactericidal activity shall

be defined.

2.1 This standard is applicable under the following conditicns:

2.1. i The metnod described is mainly intp.nded for determining thebactericidal activity of commercial formulations but it may, with yet

stronger reason, be applied to substancp.s used as active materials.

2.1.2 The product tested is intended to be used in liquid form ana shall

be water soluble or water miscible at tne test concentrations. The

solution, suspension or emulsion snail remain stable at least througnout

the test (whicn lasts a maximum of 2 h).

2.1.3 This standard is applicable only to antiseptics or disinfectants

the anti-bacterial activity of which can be neutralised by following the

procedure described in tne pretest.

2.1.4 The conditions defined in this standard prevent, as far as Possible.

foreign matter (organic material$ for example) from interfering in the

reaction between tne product under test and the bacteri a. r tis however

possible to determine, according to a simi lar procedure, the bactericidalactivity of the product in tne presence of interfering substances under

tne conditions laid down in $tandard NF T 72-170.

5

317

'IF T 72-150

2.1.5 The strains of bacteria chosen correspond to d rppresentative

range of bacterial populations.

Other Kinds of bacteri a may be used accord i ng to the same procedure.

but only in addition to the reference strains.

2.2 Using this standard, it is not possible to determine lhe

bactericid~l activity of a product at a concentration of IOOi (some

dilution IS always produced by adding the inoculum). Fur antiseptics

ùnd disinfp.ctants that can be used without dilution. it IS Possible to

determine whether these products. at a concentratiun uf 90i (10i of

inoculum) or of 50\. have a bactericidal activity complying with the

definition in Section 4.

The metnods of determining the bactericidal activity of liquid products

far whicn no neutralizing agent iS Known, åre covered by standard

NF T 72-151. The methods of determining the bactericidal, tungicidal

and sporicidal activity for the disinfection of surfaces are covered by

standard NF T 72-190. The methods of determining the bactericidal.

fungicidal and sporicidal activity of airborne disinfection processes

are covered by standard NF T 72-281.

The metnods of determining the bactericidal activity of nrooucts used

other than in i iquid fo~ wi i I be covered by future standards.

3. REFERENCES

r 72- 104

Mntiseptlcs and disinrectants - vocaoulary.

Disinfectants - Guide to interpreting standardS .orassessing tne efficiency uf disinfectants tn the medicaland hospital sectors.

Guide to interpreting standards for ~ssess i ng tneefficiency of disinfectants intne agricultural and agri-foodstuffs sectors.Disinfectants - Airborne disinfection processes - Guideto use as a function of tne results obtained accordingto the methodology 01 NF T 72-281 in the hOspital.medical. pharmceutical and cosmetic. Jgricultural,industrial and agri-foodstuffs sectors.

~IF T 72-101

r 72-102

T 72-103

- 6 -

..1 a

NF T 72-110

Pr T 72-140

NF T 72-151

NF T 72-170

NF T 72- 171

NF T 72-190

~jF T 72-281

4. DEFINITIONS

4.1 Bactericide

tlF ì2-1S0

Antiseotics cìnd disinfectants - Naminy dflU marKing ofantimlcrODlal effectiveness - Data sheet.

Antiseptics and disinfectants -' Testing of bactericìlstraiiis used for tne determination or DactericioalactivIty. .water miscihip antiseptics ana aislnfecLdilts used inliqUid form - Oetermination of bacteriCidal activity

(memorane filtration methOd).

Water miscible and neutralizaole antiseptics and dis-infectants used in 1 iquid form - Determination ofbactericidal activity in the presence af soecifi~ inter-fering substances (dilution-neutralization methOd).

Water miscible antiseptics and disinfectants used inliquid form - Determination of bacteriCidal activityin the presence of specific interfering substances

(membrane filtration method).

Antispetics and disinfectants - Water miscible contactdisinfectants used in 1 iquld form - Germ-carrier metnOd.

Oisinfp.ctants - MethOdS or diroorrie disinfection Grsurfaces - Determination of bacterlCIGal. "iingicldai anaspa ric i 0 a i act I v i t Y .

Product or process wnich has the property of killing b~cteria under

specified conditions (if the product or process is selective in relation

to a bacterial species. this shall be stated).

~.2 Determination of bactericidal activity

Determination of the capaoil i ty or otnerwi se of a proouc:S min. contact at:

r'?1uc'? 1:1

- 20°c for disinfectants and bactericidal soaps.

- 3ZoC for antiseptics.

by at least 105 times the number of living cells belongin~ to soeCltlc

strains of the following bacterial species:

Pseudomonas aeruginosa. ESCherIChia col i. Staphylococcus aureu~.

Enterococcus faecium (1). Mycor.acterium smegmatls.

(1) Formerly called "Streotococcus faecalis".

7

319

~F T 72- i 50

Spectrum 5 bactericidal activity corresponds to the use of the 5

strains mentioned above.

Spectrum 4 bacteric ida I act i v i ty corresponds to the use of the first 4strains only.

5. PRINCIPLE

5.1 Pretest

Searcn for the most suitable neutralizing agent for tne application of

this standard.

ßringing bacterial suspensions into contact with different mixtures of

the product and of a neutralizing agent for 5 min. at a tèmperature

,jefined according to tne temperature of use (200C for disinfectants and

bactericidal soaps. 320C for antiseptics otner tnan bactericidal soaps).

Counting of the colonies in these mixtures after this contact. and in a

reference series made up under the same conditions but containing an

equal volump. or sterile nistilled water instead of the product.

5.2 Actual Test

Bringing bacterial suspensions into contact for 5 min. at a specified

temperature (200 or 320C) with different concentrations of the product.

'.ounting of the colonies before and after bringing into contact, dnd inthe latter case after nputraiisation or the ~ntibacterial actl~ltV.

5.3 Performance of tne tests

The pretests leading to tne cnoice of neutral izing agent snal i be carriedout before tne actual test. As it is necessary during the final tests to

check the effectiveness of the neutralizing agent chosen. the two tests

snaIl tnen be performed simultaneously.

8

~?n

NF T 72-150

o. STRAINS

s. i Reference strains

T~e following strains snail be used tor theSe tests. it IS possible to

Obtain them from the Collection Nationale des cultures de Microorganismes

(CNCM) (FrenCh National Collection of

specialised institutes (for example:

Collectìon) (ATCC).

Type Cu i tures ). or t rom other

tne American Type Cu i ture

6.1.1 pseudomonas aeruginosa CIP A 22 (1),

6.1.2 Escnerichia coli CiP 54 127 (1) (ATCC 10536),

6.1.3 Staphylococcus aureus. Oxford strain CiP 53 154 (1) (ATCC 9 144).

6.1.4 Enterococcus faecium CIP 5855(1) (ATCC 10541).

6.1.5 Mycobacterium smegmatis crp 7 326 (1).

:.~ Verification and preservation of the strains

Tiie verification and preservation of tlie strains shall be carried out

dccording to the methods laid down in standard T 72-140.

6.3 Culture of tlie strairi~

The strains shall be used after 3 successive pricKlngs out on agar for

maintaining strains (7.2.2) under the following conditions:

Strains i nterva 1 between 2 Incubationprickings out tempera ture

6 1. 1 18 - 24 n J 7 "C = , "r.

6.1.2 18 _ 24 n 37.C = 1 .C

6.1. 18 - 24 h 37.C =: , .c

614 42 - 48 h 37 .C = 1 .c

6.1.5 42 _ 48 h 37'C = , .C

(1) Th CIP strains are distriMe by tr ColiecI() Naticrle æs Cui tures æ Microorg i 9TS - Insti tut Paster. 28 Rt OJ lbter Rr - 7~15 PfilS.

- 9 -

321

NF T 72-150

7. WATER AND CUL TURE MEO r A

7. 1 Water

The water used shall be sterile. It shall be freshly distilled water

(and not simple demineral ize~ water), free from substances whicn dre

toxic or inhibiting for tne micro-organisms. If the laboratory has

difficulties in obtaining a water of high enougn quality, water for

injectable preparations c~n be used (as defined by FrenCh Pharmacopoeial.

7.2 Cui ture media

In order to improve tne uniformity of the results, basic dehydrated

components and chemicals of analytical grade should be used for preparing

~he culture media and diluents.

7.2.1. Agar used for counting

7.2.1.1 Composi lion

~.~

Denydrated yeast extract

Tryptic casein peptone.........

Glucose....................... .Agar-rigar in powder' form.......

2. S 'J

5.0 9

1.0 9

12 to 18 9 JCCOrd-ing to Uie gel ling

propert 1 es of the '

prOduct

1,000 mlWater at above quality..........

7.2.1.2 Preparation

Jissolve the IngrPdipnts or the complete dehydrated medium in boiling

water. (f necessary, ridjust the pH so that It is 7.2~0.2 at 200 C

after sterilization (measurement carried out at 4SoC with temperùture

correct i on) .

Divide up tne medium into test tubes at the rate of 15 ml per tube or

into flasks of not more tnùn 500 ml cJpaclty at tne rrite of about halfthe volume of tne flrisks.

~terilize in the autoclave for 20 min. at 121°C ~ iUc.

- i 0 -

322

~IF 72-150

i1!"rnrp "r.nrTinn thi= test. ~ldce the suoerTuseu dflU oreviousiy cooleang~r medln in a water bath àt 40CC - 45vC.

ì .2.2 Agar for maintainina :ie strains

~ .2.2. 1 Compos i t I on

Tryptic casein peptone..........

Soya bean peptone...............

Sodium chloride.................

Agar-agar iri powder form........

OIater of -3bove qua Ii ty. . . . . . . ... .

15.0 9

5.0 g

5.0 9

12 to 18 9 accordingto the gelling proper-

ties of the product

1,000 r:l

7.2.2.2 Preparation

Proceed as for the agar usea for counting. but divide un into tUDes

~:. the rate of 8 ml per tube cr ¡rito stoppered oottlp.c; v' c;uit.ablesize. Sterilize in the autoclave. On removal from tne nutoclave. keep

the tubes in the inclined position until completely cooled.

7.3 Bacterial suspension preparation liquids

The liquidS chosen are tryptone + salt solution for strains 6.1.1 to

6.1.4. water for strain 6.1.5.

7.3.1 Composition of tne tryptone/salt solution

Trypt 1 C case i n peptone...........

Sodium chloride..................

,.ater...... ................ . .....

1.0 9

8.5 9

1.000 rr1

7.3.2 Preparation

Oissolve the Irçients in boiling water. Adjust the pH so that It iS

7.2 + 0.2 at 20°C after steriliz ation.

Divide up the liquid into 150 ml flasks, at tne rate of 100 ml per flask.

Sterilize in tne autoclave for 20 min. at 1210C i laC.

Just before preparing the odcterial suspension ailution~. put 9 ml + 0.1 ml

of liquid per tube into sterile test tubes.

- i i -

323

:-F T 72-150

7.4 Neutralizing agent

The most suitable neutralizing agent for eacn product IS Chosen according

to the results of the pretest. ~ non-restrictive list is given in

nppendi xC.

rhe neutralizing agent snail be sterile.

8. APPARA rus AND GLASSWARE

8.1 Apparatus

Ordinary laboratory equipment, Jnd in particular:

- dry heat sterilizing apparatus.

- wet heat sterilizing apparatus.

- oacteriological oven. if Possible

d homogeneous temperature of 370C

- bath of boiling water,

- water bath set to 400C - 450C,

- water bath set to ZOOC + i °c or 320C + i °C.

ventilated and non-drying. guaranteeing

a~ 1 C,

- laboratory sca les.- stirrer producing stirring of tne vortex type.

- pH meter,

- spectropnotometer fitted with a monocnromator set to the wave length

of 620 nm! 20 nm. or photometer fitted with a filter centred on this

va i ue.

- ?p.tri diShes i~reteròbly sterilized by rJdlJtion),- 5 top-wa tCh.

8.2 Glassware and small items of equipment

- Conventional lauoratory equipment (test tubes. 50 ml conical flaSks.

screwed stopper bottles),

- Z to 4 mm glass balls.

- Paral lei-sided spectropnotometry containers wi th optical path of 10 mm.

To prevent problems wIth the recycling of glass. the tubes containing tne

)roduct. t~e dilutions and micrObial suspensions and the pipettes Shall

- 12 -

324

NF T 72-150

01' nf sterile. 'JisDosaole.neutrdl glass.

8.3 Sterilization

- Dry steri lization at. not less than l700C for at least I h.- wet steri lization at 121')( + i::C for at least 20 min.

- Sterilization by filtration through membranes with 0.45 ~m pores.

9. PRETEST

9.1 Preparation of the bacterial suspensions

Remark: the suspensions of strains 6.1.1 to 6.1.4 are prepared in

liquid (7.3), whilst ttie suspensions of strain 6.1.5 are

prepared in Gis:illed water (7.1). These suspensions shall

be used within 2 !I after their preparaticn.

9.1.1 Strains 6.1.1 to 6.1.4

Sample with a scoop from ttie agar medium. respecting tne integrity of

the agar surface. strain~ 6.1.1 to 6.1.4 cultivated as in 6.3.

Spread the sample on the wet but not immersed side of a steri Ie tubecontaining tne liquid (7.3).

Then make a homogeneous suspension in tne i iquid phase by vortex type

stirring (for about 3s).

Sample aseptically d soecified volume of thiS suspension. adjust its

~DsorbencetJ 620 nm: ZJ nm by diluting wIth tne IIQula :7.31. dndadJ ust the spectrophotometer or pnotometer so as tJ obta in, in

containers of 10 mm uDtlcal path. d value between U.2 and 0.3 for

strains 6.1.1 and 6.1.2. and 0.3 - 0.45 for strains 6.1.3 and 6.1.4.

The adjusted suspension shal i contain between i x 108 and 3 x 108

cells per millilitre. It can be kept at between ¡5°C and 220C for 2 h.

- 13 -

325

NF T 72-150

9.1.2 Strain 6.1.5

The attention of ~sers IS drawn to the.fact that the handling or this

strain requires particular care. and that following of this procedure

iS essential to ensure high quality results.

Moisten the sterilized glass balls in the conical flasK with not more

than 2 or 3 drops of distilled w~ter (1.1). Sample the culture with a

scoop from the solid medium and neposlt the cells among the glass balls.

It is possible. ¿ccording to the cellular density required. to carry OUL

5 or 6 samplings. Agitate by vigorous manual sh~kirig for 2 to 3 min.Add a volume of distilled water (1.1) 4 or 5 ml more than the volume

requi red.

Agitate (2 or 3 shakes) and leave to stand for 10 min. with the

conical flask at an angle of 45°.

~ith a pipette. ~septicaiiy sample tne supernatant fluid (once only)

avoiding contact with the glass balls, transfer it into a sterile tube

and leave to settle for 10 min.

Sample the upper part and ~septically prepare tne inoculum adjusLing,

with distilled water (1.1). tne absorbence as in 9.1.1 to a.value üf

between 0.4 and 0.5.

The adjusted suspension shall contain between i x 108 and 3 x 108

cells per miililitre. It can be kept at between ISoC and 22üC for 2 h.

~.1.3 Preparation of the test suspensions (pretest)

~t the time of t~e test. ~ilute tne adJusted Suspensions in 9.1 wIth tne

liquid (7.3.1) for strains 6.1.1 to 6.1.4. and with distilled water for

strain 6.1.5. changing the pipette at each dilution,SO that they contain

2 x 103 to 6 x 103 cells per millilitre.

9.2 Counting of the bacteria in suspension 9.1.3.

Dilute 0.5 ml of suspension in 9.5 ml of liquid (7.3.1) for strdiris 6.1.1to 6.1.4. and in aistilled water (1.1) fnr strain 6.1.5.

- 14 -

326

~;¡: r ì2-\50

Mak.e a test count bY samoiina twice. 2rter s:irrin~. . -:l ~. ':dcn

the suspensions thus diluted and incorporating them r,:soectively 1:115-20 ml of agar for count i nq (ì. 2.1) supertused è t 40CC - i:svC. Aftersolidification. turn over the dishes and place tnem 11 th,: oven ¡see

9.4) .

9.3 Testing of the different neutralizing agents

9.3.1 For each of the strains. divide into two. at the rate of 4.5 m!

per tube. the different sterile neutralizing agents prepared at double

concentration. Place in the water bath at 20°C or 3ZcC (according to

5. i).

9.3.2 Add 5 ml of water (7. I) to one of the two series preoared in

9.3.1 (reference c;prips). Stir.

9.3.3 Prepare. in tne water (7.1) or in the diluent recommended by the

manufacturer for the prOduct. d prOduct di lution corresponding ~: 115

,;f the maximum bactericidal concentration specified t2r the testing Of

each strain.

9.3.4 Add to the other series prepared in 9.3.1. 5 ml of the dilution

of the product to be tested (9.3.3) (pretest series). Stir. Leave in

contact at 200C or 320C for 10 min + i min.

'L3.5 Aftpr contact, introduce 0.5 ml of the bacterial suSoensilJlI

prep~red in 9. i. 3 into each of the tubes of the reference seri es(9.3.2) and of thp pretest series (9.3.4), carefully avoiding the uoper

part of the tube sides. Start a stop-watch at the beginning of the

addition ~nd c~rry out a vortex tyoe stirring for d few secondc; :3 l~

5s) immediately ,ìfter r.r.~ '?nd of the addlti::n. C.~ave If! CcinLrlC :'1 the

water batn at 20"C + I"'C or at 32;;C + IÙC for :i min.. I miii ~xJctly.

9.3.6 At tne end of the contact time. very quickly carry out J vortex

type stirring, take (twice) 1 ml of the mixtures. transfer them

respectively into Petri dishes and incorpor~te them with is ml or super-

fused agar medium at 45°C. After solidification. turn over the dishesand place tnem in the oven immediatelv.

i 5

327

NF T ì2-1S0

9.3.1 Any experimental arrangement :an be used which ma~es I: possiule

to br i ng together:

- the product tJ be tested at a concentration of ¡/lOth at t~e maximum

bactericidal C8ncentration specified for the actual test.

- the neutralizing agent at a concentration of 90i V/v.

- 0.5 ml of bacterial suspension containing 2 to 6 x 103 cell$ ~er

mi i I iii tre.

SpifiPd bdeticiaalcaientratioo of th 100% 20 "'. 1 %

~Altr~l iZlr" aç.

I 5 ml~t20 %

I 4.5 mlI ,-ODiec:nenratiai

1 mlit.20.. 0.5 mlllt2 0/. I

9 ml I,of neral IZlng I:~t

voluæ ard caienration Prr45 ml

'AJle: ::aifftratiexi

i ßñrIñl suspesion 3

i titrating 2 to 6 x 10

I cells per millilitre

I Sterile distilled water

0.5 ml 0.5 ml 0.5 ml

4 mt

9.4 lnctIoo

Incubate the dishes corresponding to the test counts (9.2) and to those

of the reference and test mixtures (9.3.6). lid underneatn. :'ùr:

48 h at 37JC to iJC fGr strains 6.1.1 to 6.1.3.

72 h at 37JC . I;: '~r strain 6.1.4.

7 days ùt 37vC ~ ¡JC ~Jr strain 6.i.5.

9.5 Reading

9.5.1 Count t~P. colonies appearing from the dishes (9.2) dlUJ 'Jive theresult as t.hp. niimber of bacteria per miililitre; calculdte the i)verdge N.

9.5.2 Cùunt the colonies on tne dishes (9.3.6) for tne test series(nii ònd niZ) ,iM for the reference serotes (N', JM N'2). ':ùlculJte L:iecorrespond i nCJ .iverc1Cjes ii' ,1nd N'.

- 16 -

.,.,0

~¡ê T ì2-150

~.6 valldItj _. the oacterial suspension ana c~oice :r neutra~izin~agent

j.6.1 CheCK that the result U obtained in 9.5.l corresponds to cì tpst

suspension titrating from 2 x i03 ':J'& x 103 cells per millilitrp..

9.6.2 Note the neutralizing agents wnich. for eacn :;f ttie sërains. Me

such that at least 50\ of the bacterial cel is are protected by the

neutralizing agent from thE' cìntibacterial action 01 the ~ruduct. l.~.:

n i), 0.5 N'.

Check. that N' is little different from N. showing tnat the neutralizinCJ

agent has no effect on tne bacteri a i suspens i on.

9.6.3 For each strùin choose. among the neutralizing dyents tested. the

mas t act i ve one and note the product concentrat i on I n tIie tubes 9.3.3for which this result is obtained.

:f necessary. different neutraltzing agentS,but ones which cìre sultaDlf

ror eacl, strain,will be able to De used tor the actual test.

10. ACTUAL TEST

The determination of the bactericidal activity consists or carrying out

the actual test as indicated below. and of simultaneously reppating tne

pretest val idating the neutral izing agent.

10.1 Prepùration of the bacterial suspensions (test suspension)

Prepare tne bacterial suspensions at tne time of tne test as inaieated

in 9. i . 1 and 9. i .2.

The adjusted suspensions titrating from 1 x ioe to 3 x IO~ cells per

~illilitre are used as the test suspension. Kept èt between ISOC and

22°C, they shal i be used wi thin 2 h after tnei r preparat i on.

10.2 Counting of tne bacteria in the. test suspension

10.2.1 using the adjusted suspensions prepared in 10. i. mcìKP di lutionsup to 10-6 by transferring i ml of this suspension, then successivp

~ilutions inùo liS containing 9 ml of bacterial suspension preparation

- ì 7 -

329

'IF T :2-150

liquid (7.3) for strJins G.1.1 to 6.i..¡, dnd distilled water (7.:)

fnr strain 6.1.5.

:0.2.2 After vortex t!pe stirring. maKe 2 samples or i ~i. ~r~nsfer ~dcn

Jf them into a Petri disn.then incorporate them ImmedlatPoIY into 15 ~:

20 ml of agar medium (7.2) superfused at 40-450C. Aftpr ~JlidirlCJtion.

incubate the úlShes as in 10.5.

-410.2.3 From the 10 Jilution. prepare by 1/5 dilution in the liquia(1.3) for strains 6.1.1 to 6.1.4 or in water for strains 6.1.5. jsuspension titrating from 2 to b x 103 cells per mi11ilitrp intpnded f)r

validation of the neutralizing agent.

10.3 Validation of the neutralizing agent

;':1.3.1 For each or Cie strains. divide the neutralizing dqent :::iosen;11 9.6.3 and preparea with double concentration (200'%) into two, .It therate of 4.5 ml per tube. Place in tne water batn at 20° or 320C.

lO.3.2 Prepare as many 4.5 ml portions as there are product dilutions

to be tested. of the neutral izing agent as in 10.3.1. Add 4.5 ml of

water (1.1) to the tubes tnus prepared. Agitate and leave in the water

~atn these tubes intended for the product test.

10.3.3 Into botn tubes (10.3.1). add respectively 5 ml of water (7.1)(reference neutralizing agent) and 5 ml of thPo product dilution corres-ponding to 1/5 of the maximum bactericidal concentration specified fer

~he testing or eacn strain (validation of thp nputr~iizin0 ~~ent).

\qitate and l'?dve in contact for !O min. I 111n ,iI 2Jve,r, ;:v,.

10.3.4. After cantelct. ¡Jut into these 2 tubes,colrefully:voiciiriq r.ilE'upper part of the sides of the Lube. 0.5 ml of J bacterial ~ilution

(10.2.3) titrating from 2 to 6 x 103 cells/mi. Start up ù stop-wdtCh

elt the beginning of the addition and carry out d vortex type stirring

for el few seconds (3 t~ 5 s) immediately the addition hJS finisned.

Leave in contac tin tne wa ter lJa ttl at 200C 1°C ùr .n 32;'( . I ,ji: r or5 min. '2xactly.

ie

NF T 72-150

10.3.5 At tne end of the contact time. and vpry Quickly, carry Out

a vortex type stirring, sample (twice) i ml of the mixtures. transFer

them into Petri dishes and incorDorate with 15 - 20 ml of superfused

agar medium. After solidifi:ation. turn over tne oishes and incubate

them as in lO.S.

10.4 Product tests and counting of tlie surviving bactcrld

10.4.1 According to the presumed degree of activity of the product

under test. prepare for eacn bacterial strain, in water (7.1) or in

the d i 1 uent recommnded by the manufacturer, a range of productdilutions at twice the concentration of that whicn it is wIShed actually

to bring into contact with the bacteria, which can, for example. be

cnosen from the following table:

Final cc:eitrationsin 1. m/v or V/V .

90 7.5 0.75 0.07575 5 0.5 0.0550 2.5 0.25 0.02525 1

I

0.1 0.0110

Ieic.

Place these dilutions in tne water bath at 200C or 320C.

10.4.2 Put 1 ml of the adJusted bacterial suspension (10. i) into dsteri Ie tube. carefully avoiding the upper part of the s Ides of thø.tUbe. After adding 4 ml of water (7. i), pi ace tne tube i n the water oathat 20°c t IÜC or 320C t laC for at least 5 min. until stabilization at

tne temperature whiCh shal i be Checked. Prepare in tnis manner as many

tubes as there are product dilutions to be tested. and proceed similarly

tor eaen of the test strains.

10.4.3 Put 5 ml of eacn of the product dilutions into the tube containing

the bacterial suspension and the distilled water. Start up a stop-watch

dl tlie beginning of thc addition. ilnd carry out a vortex type stirring for

19

331

,'iF T ì2-150

a few seconds (3 to 5 s) irrediately the addition has finIShed. Leave

in contact in the water batn at 20°C ~ 1°C or at 320C ~ 1°C for 5 min.

exactly.

10.4.4 At the end of the contact time, and very quickly. carry out Jvortex type stirring. take L ml of mixture and transfer i t into 9 mlof neutralizing agent (ID.3.2) previously placed in the water bath at

2UoC + 1°C or 32°C ~ 1°C. For this test to be valid, the stop-watch

shall not snow a time of more than 5 min lOs at the end of the operation.

10.4.5 10 min. after the transfer into the neutral izing agent, take two

I ml samples of the preparation for the counting of the surviving

bacteria. Proceed as in 10.2.2.

10.4.6 At the end of the test. measure !he pH in the tubes where the

bacteria have remained in contact for 5 min. wi th the product at tne

maximum and minimum concentrations.

10.5 Incubation

Incubate tne dishes corresponding to the countings before contact with

the product (10.2.3) ~nd after contact with tne latter (10.3.5), lid

underneath. for:

48 Ii at 370C ¡. laC for strainS 6.1.1 to 6.1.3

12 h at 310C + 1°C for strain 6.1.40- J

7 days at 31 C ~ i C for strain 6.1.5.

10.6 Read i ng

10.6. i Count the colonies appearing from the dishes (10.2.2 and 10.3.5

(n!f~ naral izing agent)) and note the average resul ts Nand N' of the

2 tests performed respectively from the dilutions in tne bacterial

suspension preparation liquid or in distilled water according to'the strain,

and in neutra liz i ng agent. Refer th i s resu i t to the number of bacteri ain 1 ml of inoculum in ordèr to cneck that It actually contained a

number of cells per miililitre of between 1 x 108 and 3 ~ 108, N' having

to be i i ttle dl fferent from N.

- 20 -

.,., .,

NF i ì2-ISO

!0.ó.2 Count ttie coionies dPpearlng from tne disnes \ 10.4.5) annnote the average result n of ttie 2 tests performed for each dilution

J f the produc t.

10.6.3 Count the colonies appearing from the dishes (10.3.5) (vallda-~.ion of the neutralizing agent) and note Uie average result n' of tne2 tests performed for eacn dilution of Uie product.

11. EXPRESS I ON OF RESULTS

11.1 Check that n' (10.6.3) iS not less than 0.5 N' (10.6.l). If this

condition is not fulfilled. the neutralizing agent iS not validated and

the test shal I be rejected.

11.2 For eacn of the bacterial strains, look for the prOduct concentra-

:ions for wnich:

n( 10.6.2) 15 not more ttian: N'

1õ

whicti corresponds to a x 105 reduction in the number of bacterial cells

and defines the bactericidal activity.

11.3 In cases where no neutralizing agent satisfying the tests can befound, standard NF T 12-151 shall be applied.

11.4 Spectrum 4 bactericidal activity characterises products for which

the test shows that conditions 11.1 and 11.2 are satisfied for strains

ó. i . i to 6. i. 4.

)pectrum ~ bactericidal activity cnaracterises prOducts for wniCh the

:est snows that conditions ll.1 and 11.2 are satisfied for 'strains

õ . i. I to 6. i. 5 .

12. REPORT

The report snail refer to tnis standard. specifying spectrum 4 or

spectrum 5 as the case may be. It shall give the result obtained for

¿aCh of the strains and state ~Il the requisite details for t~e full

21

333

~F T 72 -150

identification of the product sample subjected to the test. the nature

and tne initial concentration of, the active substance(s) when given by

tne manufacturer. and the pH l n the tubes in wh (ch the product ~as

tested at the minimum and maximum concentrations.

It shall give all the procedural details for whicn the standard leaves

a possibility of choice (for example. tne source of the strains: CNCM or

ATCC). all the procedural rletails not laid down in the standard and any

incidents tnat mignt h~ve affected the resul ts.

It shall indicate the neutralizing agent(s) used and detail the results

which allowed this choice to be made.

In order to refer to this standar~. the test report shall contain all the

information mentioned in Appendix A or B.

22

11~

'IF i ;-.: - i 50

:"PPEND I X

: formi nq an 1 nteqra 1 part cf the

3tanaard)

LAYOUT ANO COMPILATION EXAMPLE OF A REPORT FOR DETERMINING THE3ACTERrCl0AL ACTIVITY (Spectrum 5)

(diluticn - neutraì isation method)

A.l Laboratory whicn carried out tne test

A.2 Complete identification of the sample

Name of the product...............

Oatch No..........................

Manut acturer. . . . . . . . . .. . . . . . . . . . . .

Date of acceptance at the laboratory....

Storage conditIons..................

z

Y

those of themanuf acturer

;inJIYsls period.....................

Active substance(s) and concentration(s)

(optional)......................... . not indicated

A.3 Experimental conditions

A.3.1

A. 3.2

Test temperature..............

P roduc t d iI uen t recommended by the

manuf acturer . . . . . . . . . . . . . . . . . .

A.3.3 Product diluent used..........

20°C .. ¡ uC

disti lIed water


A.4.1 COmposlticn................... I ec i tn i n

A.4.2 Concentration.................. 3% (ii/V)

A.4.3 Mettmof sterilization.........

- 23 -

335

';F "2-150

.\.5 :ir~tes i: s

';.5.1 :: xper \l1en ta 1 ;~su I ts

?rct c:rPlrrlt i ens in Strains. sae colleciai rll( 'I .,'1. n(re test ~iJ. 1:1 ti coi lecien

196 3i IV/VI 195 ii 19521 19521

5 "'. P,.udomon.s ..,uglnou ; 65 160 95CIPA ii

5 "'. Eieh."ehi. cali 2.16 :58 1 87CIP 54 127

5 "'. , St.øhvioeoeeus .u'.Uf 122 110 72

I CIP 53154~ ~u

I Enr.,ococeuf I..cium

164 154 161CIP 5855

s ~o i Mvcob.et."um im.gm.tls 121 109 87! CIP 7 326

A.5.2 MdxlmUm nactericidai concentrations of the proauct for wnich

the validity or the neutralizing agent is verified

Pseudomonas ~eruginosa.............

Stapnlyococcus àureus..............

Escnerichia CJli...................

Enterococcus raecium...............

~ycooac ter i um smegma tis. . . . . . . . . . . .

5: (V/V)

51 (V/V)

51 (V/V)

5i (V/V)

51 (V/V)

¿4

336

NF T 72-150

~.:i ¡'.:tual 7.est

,~.6.i ~xDerimental results

I . i

IN N. " n (10621

Strains. :.ree (1022) 11035) i 10351 ¡ Ccraticr in pH;:ollecicr ard rb.

val i. t:u.r:rctã iV/V)

in tr collecicr in ccn Wi tn tlbaria min

Ima.

100""3000.5 10.751 I 2.5 ¡ 5.0 I i1 0.5 % 5 %

ii

I IPseudomon~s ~eruginos~ i 80 212 222 + 56 30 1 a 51 5 7

CIP A 22 Ii

E scherlchi. coli I 196 208 215 .. 64 19 1 a 53 59CIP 54 127

I

i

Sr~phvlococcus ~ureus 200 ~" ., 233 .; .; 32 4

I

a 5.5 56

I

~~4CIP 53 154 I

I

I

;

i I

i

Enrerococcus I,Jeclum 262 28!) 292 + - + -I

1 52 58Cio 5 855

I

ii

I

i i i

Mvcob~cre"um smegmaris i 60 188 i 96 ~ i-

i- ¡ - a 5 3 59

CIP 7 326

Concentration~ for whiCh n ~N'

TI Jre bactericidal

if n i ~ 0.5 N' and if N ~:,. N i .

A.6.2 DrOduct. ... possesses a spectrum 5 bactericidal activity at20°C ~r 32:C (see A.3.!) JCCOrding to standard NF T 12-150 (1981).

25

337

'IF T 72-150

APPEND I X a

(forming dri Ir1tegral part cf ':he s::anoarcJI

~AYOUT ANU COMPILATION EXAMPLE OF A REPORT FOR DETERMINING irlE8ACTERICIDAL ACTIVITY (Spectrum 4)

(dilution - neutralisation method)

B.l Laboratory wnich carried out the test

B.2 Complete identification of the sdmnle

Name of the prOduc t. . . . . . . . . . . . . . . . . . . . . . . : ZBatcn No..................................

Manuf acturer. .. . .. . . .. . . . " .. .. ... . . . .. .. .

Date of dcceptance ~: l~ie I Jborator'l. .. .. .Storage conditioI1S........................

y

1980-04- 3u

thOse c. r' :.~emanufacturer

Analysis period...........................

Active substance(s) and concentrñtion(s)

(optIonal)............................... .

1980-05-15

not indicated

8.3 Experimental conditions

ß.3.1 Test temperature

B.3.2 Bacterial suspension preparation I ¡quid usP. during

the tests

3.4 Neutraluirig agent

B.4.1 Composition......................... lec i t:iin

B.4.2 Concentr~tion....................... 3'1 (m/V)

ß.4.3 Methud of steri 1 izJtton.............

26

....0

NF T ì2-15U

-TULe~ L:.

3.:i. i ¿xperimenta L resu Its

:JroouCt CJncentrat ions Strains. souce collecion ana N N n

; 1 tiH? tes t tt. in tt collecion

('J 6.31 (V IVI 195 i J 1~52) i'J 5 2)

50/. iv/V) Ps.udomon.s ~~,uginos~ 165 160 95

CIP A 22

5 % IV/VI Esch.rrch,. colt 246 258 187

CIP 54127

5 % IV/VI St.phy/ococcus ~u,~us 122 110 72

CIP 53 154

5 '!.IV/Vi Enttt,ococcus f~ttcium , 64 154 161

CIP 5 855

13.5.:: r~aximum Dùctericidal concentrations at the product for wlllCh

the validity or the neutralizing agent iS verified

Pseudomonas aeruginosa................

Staonylococcus aureus.................

Escherichia col ¡ ..................~..

Enterococcus faecium..................

5i (V/V)

5i (V/V)

5i (V/V)

5i (V/V)

21

339

'IF r ì2-lS0

3.6 Actual t:?st

B.6.t Experimental results

Strains. sa.ræ N N' n n (10 621

collecicr ar ~. (102.21 110.3.51 110351 Ccnaitraticr in pH

in tJ collecicr v".\æ teti ~rc9' (V IV ) inccr Wi th tr

bdteria min m..iooarOO

0.5 10.75 2.5 15.0, 0.5'" 5 '" I

Puudomon.. ..ru9,"or. I iao i 12 I Z22

I

.. 56

I

i 30 0 5.1 57 ICIP A 22

I I

IE seh.",:hi. eoli 196 Z08

I215

I

.. 64 19 1 0 53 59CIP 54 127

I;i

S 1."h.,loi:øi:t:u. .ur.us zooI

zzi i 233 . ~ 32 4 0 55 56 ¡

I ICIP 53 154 ;

i ¡i

I

! E"I.roeoi:i:u. I..i:,um ¡i

I

:52 ; :85 ! 292 -I

-I

- - 1 52 58

i CIP 5 a55¡

I ¡ I Ii i :

N'Concentrations for whicn n ~ Tr are bactericidal

ifni ¿0.5N' and ifN&N'.

ß.6.2 Product.... possesses a spectrum 4 bactericidal activity at

zoCe or 3Z0e according to stanaard NF T 12-150 (1987).

28

NF T ìZ-150

APPEND I X C

(not form! ng an i ntegra 1 nart of thestandard)

NON-RESTRICTIVE LIST OF ANTIBACTERIAL

ACTIVITY NEUTRALIZING AGENTS

ror carDOllC products

- freSh egg yolk diluted to si or to O.5t (V/V)

- preparation containing 3t of polysorbate 80 (1) V/V

O.4i of lauryl sodium sulphate (m/V)

O.3t of lecitnin (m/V)

- orepdration containing

- preparation containing


For ù I denydes

- preparation containinq

5~ of freSh egg YOlk (V/V)

4\ of polysorbate 80 (1) (V/V)

7t of ethylene oxide condensate over fatty

alconol (2) (m/V)

2i of lecithin (m/V)

4i of Dolysorbate 80 (1) (V/V)

4t of ethylene oxide condensa~e over fattyalconol (~) (m/V)

4i of lecitnin (m/V)

3i of polysorbate 80 (1) (V/V)

0.3i of lecitnin (m/V)

O.li of L-histidire (m/V)

(1) K. by th trac: Ti, 00. of anlytical and ro-hØ'lys qui i ty.

(2) For cxdIle ii.orol i-.

29

341

:¡F i 72-150

- T.H.L.C 3~ Tween

3~ sapon i ne

0.1: his tid i ne0.11. cysteine

For quarternary ammoniums

- fresh egg yolk diluted to 5% (V/V)

- preparation containing 5% of fresn egg yolk (V/V)

3l of pol ysorbate 80 (1) (V/V L

- preparation containing 3% of polysorbate 80 (1) (V/V)

0.31 of lecithin (m/Vl

- preparation containing 3l of etnylene Oxide condensate over tatty

alcohol (2) (m/V)

2~ of lecithin (m/vi

- preoaration containing :01 Ot a 50 ~g/ml pnosDnollOld emulsion

(m/V L

For organo-mercuri a i s and products conta i n i ng other heavy meta i s

- sodium thioglycolate at 0.051 or 0.51 (m/V)

- L-cysteine at 0.081 or 0.151 (m/V)

- thiomalic acid at 0.075i (V/V) adJusted to pH 7 by sodium nydroxidp..

Fur halogenated derivatives (hypocnlorite. cnloramine T. iodopnores, etc.)

- sodium ttiiosulptiate at 0.5i (m/Vl

For perox i des- (~atalase

- peroxidase jFor Doth tnese enzymes. Jnp. unIt cJtalvses the

0eccmposition or 1.4 gram~molecule of hyorogenperoxide per minute eH 25"C ,n pH 7.

(1) Kso, by Uie traæriæe: T.. 00. uf analytical ard rc-nyarolysec qual ity.

(2) Fur exêJle LLDrol i,.

30

w ~ w

..

9 1

)O

il I i

5 ----

,\ '

\. IOJ:.:11. rnil ''\

121.

6.-

\ '\

, U

i 0

IN 1M I ~ I

i u I

i ~ I

I N I

I :; I

i !: i

i E i

o I I I I I I I I

L C C/5

~ B

JClL

riJI

sU

SpeS

iUl l

iqui

d (7

.3)

= r-t1Xmrn speified bactericid::l cal,-entratim

= 1

/5 "

\ixim

rn S

i~'e

(ifie

d ba

::ter

icic

.i1 c

cnce

ntra

tiCl Pr

etes

t

1\\ '.

0.5

rnl ---

-

'\ 0 ,.s

.9 o

J '\

'?/

S \,

-

8-~

0(1

\ __

%....-. ~ 1-

r.

--~

092

_..

~~0

~ 1 Q

iI

IIl

I

1I

__, r

r i

0I u

I9~

...I 0

i :: I

i :;

1

I U I

i ~ I

0I N

Il ~

I i; I

~z

I £ I

"lI

91 Q

I

-,r¡

-J N

0,

-"""

/ttl

- LY

'9~

..0

10. _

_--

-

1110)'( 108

'''''/mll Ilto)X 104coll",iill

5 11.1

Pro

iJC

l __.

__C/519 3 3) 9 34

/~l"

\i.~ ../,

./ ')

. ag:t/ ~

latr

aliii

o;(OCle con- __q.Srrl

cenr

atim

) 9?

ì--

50'1

_.-

-9--

32Disiilled w3ter

:--~

-- \ \ 0 úl \'

dv\ ?

d\ / \ \ \ \ \ '\

';F T ì2-IS0

o10.4 - U 10'¡ U

.t: - tD

i~\~~.i~-~1022 --

10.1

(1 toJ X 10ltcllVmii (ItcJX 104tellVmll

Disti lled wate

", V. .s/0' ."... "//'"5ml'-

i

".89\~~\ ~\ ~

\ 'ô I 11j r:, ,I ~~!:-\ -- : tt : i~~:~n.i'; N I Js nN ..i? I'" i 2I ~ 0 \'" i .'.

i .. i. ~ I u.

.\- 0I'; INi:: i 1i :¡ I 1 i .': i

¡~ ~---o-:~:-Oi I i II

10.J: tes

p (e/51S ml

10.3.3-

~airal izirg ~t(áJle ccnation)

Calecc:tionPrc

l"I . I..

sml_U_:r.U I~/ :ã

eo~X IØ.. eo...' ,0' 10...3

õ\-,. !N

,~yG~'J,0. . N"

?Õ.. 0vJ~G10.3.3

Test 10.4

lml1044

i l' iI:: I

-~_:~9ml I:i i

10:-0i

ø8nl, i(

,o..~ n,'0 ~,.~

S

10.4.5

Disti lIed water

L = Baridl SUs¡Sion I ¡QUId (1.3)

C = Mainu speified bateicidal ccnentrationCiS = lIS Mainu spiflPC bateicidal icn91raticn

Actual test and simultaneous reoetition ot tr.e pretest

;2

u....uc:OJ...u::

:i IIUZ Inti...0:c. ..

uti c::i OJE- w

c.:io c:0: ...c. ..o IIti c:E- ...~ 0'...~U) wZ 0~0: OJl: ..

.u

Af~.~/LfRENCH STANDARD NfT72-170

November 1988

Water miscible and nCutralizable anriseptics

and disinfectants uzcd in liquid form

Determination of bactericidal activiry in the

presence of specific interfering substances

Dilution-neutralization method

f : Antiseptiques et désinfectants utilisés

à l'état liquide, miscibles à l'eau et

neutralisables - Détermination de l'acti-

v i té bactéricide en présence de substancesinterférentes de référence - méthode par

d i 1 ut ion-neutral isat ion.

French standard certified by decision of the

Director General of Afnor, un 5 october 1988

to take effect on 5 november 1988.

Replaces the standard of the same number of

March 1981.

Cor respondence At the date of publication of this document

there is no international standard on this

subject.:i..c:o Analysis This standard makes it possible to assess and

compare the quality of products used as

antiseptics and disinfectants. investigation

of the activity is performed in the laboratory

(in vitro) under defined conditions and in the

presence of interfering substances : using the

dilution-neutralization method it makes it

possible to determine a reduction of at

least 105 times the number of living cells

belonging to the selected strains of bacter ia.

Published and distr ibuted by 1 i Association Prançaise deNormalisation (AFNOR), Tour Europe, cedex 7, 92080 Paris laDéfense - Tél : (1) 42.91.55.55

C9 Afnor 1989

372

NF' T 72-170

Desc r i ptor s I nte r nat iona 1 Techn ica 1 Thesau rus : Disinfectdti:,liquids, water miscible products, determination,

bactericidal activity, culture media,

composition: property, dilution, neutr~liiing

Modifications In respect of the standard of March 1981, the

procedure has been improved, two test alter-

natives using either 5 strains (spectrum 5)

or 4 strains (spectrum 4) have been included,

and the expression of resul ts has been

amended. A distinction has be~ made between

the basic standard bearing this number and

the appl ication standard intended to determine

concentrations for application in variable

conditions.

Corrections

,

373

Nf T 72 - i 70ANTISEPTICS AND DISINFECTANTS

BACTERICIDAL ACTIVITY

Members of the standards committee responsible for preparing ~h\s

document

President: Professor CREMIEUX - F~culty of Pharmacy - Marse~iip

Secretary: Mme BESSON - AFNOR

M.

M.

MUeM.

M.

Mme

M.

MlleMIleMIleM.

Mme

M.

Mme

M.

Mme

Mme

M.

MIleMUe

BARDONESCHI

BEERENS

Henkel France

UER de Pharmacie de Lille

CHALE

CHANTEFORT

AEEISO

Lab National de la Santé de

MontpellierLonza FranceDiversey France SA

Pharmacie Centrale des Hôpitaux è~

ParisSICCA

Institut d'Hygiène de Strasbourç

Ch Synd Nat de l' eau de javelFacultè de Médecine de Clermont-

FerrandExpertLaboratoire MIDAC Lilte

ANIOS Lille

Lab National Médicaments Vétér i~;ire

de Fougères

Laboratoire d'Hygiène de la Vii:~ de

ParisRhone Poulenc Santé

SANOFI RechercheHOpital Edouard Herriot de Lyon

BOF Médical SA

CHARCOSSET

CHEVRETON

DARBORD

GAUTHIER

HEITZ

JACQUET

JOLY

LELLOUCHE

LEPAGE

LETARTRE

MARIS

MORIN

POMMEYROL

PONS

REVEROY

VOCE

3

374

NF T 72-170

fOREWORD

This standard, which has been prepared on the same principles as

NF T 72-150, to which iL refers, is intended:

- to investigate whether or not a liquid product which is intended as an

antiseptic or disinfectant, and has satisfied the tests given

1n Nf T 72-150 or NF T 72-151, has a spectrum 4 or spectrum 5

bactericidal activity under the laboratory conditions defined in

the standard.

- to compare various products under reproducible conditions

Applied in addition to the aforementioned standards, it makes it

possible to compare bacter i cidal concentrat ions wi thout and wi ththe presence of interfering substances liable to modify the

bactericidal activity of the product. The interfering substances

proposed by this standard are frequently encountered when the

antiseptic or disinfectant is applied in practice, namely:

- mineral ions (from hard water or buffer systems)

- 3 protein and a cellular hydrolyzate

Another standard is being prepared for situations where these

substances on the one hand and/or the strains and test condi t ionson the other hand are not representative of actual conditions inpractice.In the absence of other application standards, the concentrations

defined in standard T 72-170 may be used as application

concentrations for uses involving contact times and temperatures

that are similar to the experiment conditions in the test.

4

375

NF' T 72-170

CONTENTS

1 SCOPE

2 FIELD OF' APPLICATION

3 REFERENCES

4 DEFINITIONS4.1 Bactericide

4.2 Determination of bactericidal activity in the presence of

specific interfering sub~tanccs

5 PRINCIPLE5.1 preliminary tests

5.2 Main test

5.3 Test procedure

6 PHYLA6.1 Specific phyla

6.2 Control and preservation of phyla

6.3 Cultivation of phyla

7 WATER AND CULTURE MEDIA

7.1 Water7.2 Culture media

7.3 Bacterial suspension liquids


7.5 Specific interfering substances

8 APPARATUS AND GLASSWARE

8.1 Apparatus

8.2 Glassware and small equipment

8.3 Sterilization

5

376

9

9.19.29. J

9.49.5

10

10.110.210.310.4

11

12

13

NF T 72-17Q

PRELIMINARY TEST

Preparation of bacterial suspensions

Bacteria count in ~uspension 9.1.3

Verification of neutralization in the presence of

interfering substances

Readingvalidity of the bacterial suspension and selection o(

neutralizing agent

MAIN TEST

Preparation of the main tes~ suspension

Bacter ia count in the test suspension

Testing the product

Reading

INTERPRETATION

EXPRESSION OF RESULTS

TEST REPORT

APPENDIX A

A.PPENDIX B

A.PPENDIX C

6

377

NF T 72-170

1 SCOPE

The purpose oE this standard 1S to supplement the methods for

determining bactericidal activity with the dilution-

neutralization method (NF T 72-150) or the membrane filtration

method (NF T 72-151), by defining supplementary test conditions

characterized by the preseAce of specific interfering substànces

which are liable to modify the bactericidal activity of the

product. It makes it possible to define w~ether or not a product

is a spectrum 4 or spectrum 5 bactericide in the presence of the

interfering substance being studied, or, in the absence of other

standards, to define application concent~ations for uses

involving contact times and temperatu res imi lar to the expe r iment

conditions in the test.

2 FIELD OF APPLICATION

2.1 This standard applies to products that satisfy the tests

specified in NF T 72-150 or NF T 72-151 under the following

cond i t ions:

2.1.1 The method described is mainly intended to determine the

bactericidal activity of commercial formulations, but it may, a

fortiori, be applied to substances used as active ingredients.

2.1.2 The product tested is intended to be used in the liquid

form and shall be soluble or miscible in water at the

concentrations used in the test. During the test (which lasts

for a maximum of 2 hours) the product shall not form a

precipitate in the presence of the interfering substance used and

the solutions or, where applicable, the suspensions or emulsions

obtained in processing shall be stable. In the case of

flocculation, the standard does not apply to the interfering

substance in question.

7

378

NF T 72-170

2.1.3 This standard only applies to antiseptics or disinfectants

whose antibacterial activity may be neutralized following the

procedure described in the preliminary test. If this is not

possible, use NF T 72-171.

2.1.4 The choice between the different interfering substances is

made according to the practical use intended for the product.

The interfering substances u~ed are:

- bovine albumin, as a protein,

- yeast extract, as a cellular hydrolyzate,

- a specific solution of calcium, and a specific solution of

calcium and magnesium, as waters of standardized hardness, (l)

- 2 buffer solutions corresponding to an acid medium and an

alkal ine medi um.

Reference mixtures of these substances are proposed in this

standard. For any other interfering substance or any other

mixture refer to Standard NF T 72-300 (in preparation).

2.1.5 The bacterial scraL~~ selected correspond to a

representati ve range of bacter ial popu lat ions.

Other species of bacteria may be used following. the same

procedure, but only as a supplement to the specific strain~.

2.2 This standard cannot be used to determine the bactericidal

activity of a product at a concentration of 100\ (a certain

amount of dilution is always necessary to supply the inoculum and

the interfering substance). In the case of antiseptics and

(1) Standard FIL lOF 44 (1967)

8

379

NF T 72-170

disinfectants that can be used without dilution, it may be

determined whether, ùt a concentration o( 50' (40' interfering

substance and 10\ inoculum), these products have an activity in

accordance with the definition given in Section 4.

Note: test concentrations greater than 50' may not be studied in

accordance with this standard. If this is necessary, the

tests may be performed on a hatch of antiseptic or

disinfectant that has been specially manufactured at twice

the concen trat ion a f the commerc ia 1 fa rmu lat ion.

3 REFERENCES

NF T 72-101 Antiseptics and disinfectants - vocabulary

T 72-102 oisinfectants - Guide tointerpreting standards to determine the efficiency

of disinfectants in the hospital and medical sectors

T 72-103 Disinfectants - Guide tointerpreting standards to determine the efficiency

of disinfectants in the agricultural and agro-food

sectors

T 72-104 Disinfectants - procedures fordisinfecting surfaces by air - Guide to use as a

function of results obtained in accordance with the

methodology of NF T 72-281 in the hospital, medical,

pharmaceut ical and cos~etic, agricultural,

industrial and agro-food sectors

NF T 72-110 Antiseptics and disinfectants - Denomination and

marking anti-microbe efficiency - Specification

sheet

9

380

NF T 72-170

T 72-140 Antiseptics a'nd disinfectants - Preservation and

control of bacLerial strains used to determinebactericidal activity

NF T 72-150 Water miscible and neutralizable antise¡Jtics anddisinfectants used in the liquid form -

Determination of bactericidal activity - dilution-

neutralization method

NF T 72-151 Water miscible antiseptics and disinfectants used in

liquid form - Determination of bactericidal activity

- Membrane f i 1 tra tion method

NF T 72-171 ~ater miscible antiseptics and disinfectants used inliquid form - Determination of bactericidal activity

l.n the presence of specific interfering suostances '-Membrane filtration method

NF T 72-190 Water miscible contact disinfectants used in liquid

form - germ carrier method - Determination of

bacter icidal, fungicidal, spor icidal act i v ity.

4 DEFINI TIONS

4.1 Bactericide

Product or process that has the property of killing bacteria

under defined conditions (if the product or process is selective,

in respect of a species of bacteria, this shall be specified).

4.2 Determination of bactericidal activity in the presence of

specific interfering substances

Determination of a products's ability or inability, in the

presence of specific interfering substances and with 5 minutes'

contact at:+ 20°C for disinfectants and bactericidal soaps,

+ 32°C for antiseptics,

to reduce, by at least 105 times, the number of living cells

10

1R1

NP T 72-170

belonging to netermined str~,sof the follo~ing species of

bacteria:

Pseudomonas ae rug i nosa, Escher i ch i a col i, Staphy lococcus aur eus,

Enterococcus faecium, Mycobacterium smegmatis.

Spectrum 5 bactericidal activity corresponds to application of

the 5 st~~ ment ioned above.

Spectrum 4 bactericidal activity corresponds to application of

the first 4 only.

5 PRINCIPLE

5.1 Preliminary tests

The interfering substances and the product to be tested are

brought into contact with each other for 5 minutes at 200C or

32°C. After transferring to the neutralizing agent, contact ismaintained for 10 minutes. Bacterial cells are added and contact

is maintained for 5 minutes at 20°C or 32°C.

The colonies in this mixture are counted as are the colonies in

the 2 references obtained by bringing bacterial cells into

contact with the interfering substance at 200C or 320C for 5

minutes and putting bacterial cells into contact with the

neutralizing agent at 20°C or 320C for 10 minutes.

5.2 The main test

Suspensions of bacter ia are brought into contact wi th theinterfering substance for 5 minutes at 200e or 32°C. The product

is added, at various concentrations, for 5 minutes + 5 seconds.

The bacteria are counted before and after contact and in the

latter case after neutralization of the antibacterial activity.

11

382

NF T 72-1705.3 Performance of the tests

The preliminary tests leading to selection of the neutralizingagent shall be performed before the main test. ~s it ia

necessary during the final tests to control the effectiveness of

the neutralizing agent used, the 2 tests shall then be pe~formed

simultaneously.

6 STRAINS6.1 Specific S~(ains

The following strains shall be used for these tests. They may be

obtained from the Collection Nationale de Cultures de

Microorganismes (CNCM) (National Collection of Microorganism

Cultures) or from other specialized organizationa (for example

the American Type Culture Collection) (ATCC).

6.1.16.1.2

Pseudomonas aeruginosa

Escherichia coli

6.1. 4 Enterococcus faecium

CLP A 22 (1)CLP 54127 (1)(ATTC 10 536)ClP 53154 (1)(ATCC 9 144)ClP 58 55 (1)(ATCC 10 541)CIP 7 326 (1)

6.1.3 Staphylococcus au reus souche Oxford

6.1.5 Mycobacterium smegmatis

6.2 Control and preservation of the strains

Control and preservation shall be per formed in accordance wi th

the conditions defined in Standard NF T 72-140.

(II CIP strains are distributed by the Collection Nationale desCultures de Microorganismes - Institut Pasteur, 25 rue duDocteur Roux - 75014 Paris - FRANCE

12

383

NFT72-l70

6.3 Cultivation oE st rainsThe s trairs s hall he use d a E t e r J s u c c e ~!J 1 vet r ;1 n s p 1 ant son t () the

gelose for prp.5p.rvin~ ~r~ns (7.2.2) under the followingconditions:

~trc:ins Tim bci:en Incubation2 transplants temprature

(, . I . I IB to ,J4 hrs J 7 .c + I . r.

IB to 24 hrs J 7 . C-

.cti. 1. 2 .¡ I

()o I . J IBt021,hrs J I . r. :; l .C(,. I ." 42 to 4H hrs J 7 .c :; 1 .CÚ. L. ~ 42 La I,ll hrs 17 °c '+ i .c

. -

7 WATER AND CULTURE MEDIA

7.1 Water

The water used shall be sterile. It shall be freshly distilled

water (and not simply demineralized water), free of any

substances that are toxic or inhibiting to the microorganisms.

If the laboratory has difficulties in obtaining distil led water

of ' sufficient quality, it may use water for injectable

preparations (defined by the French Pharmacopoe ia)

7.2 Culture media

To improve the uniformity of the results, it is recommended that

dehydrated basic constituents and analytical grade ,chemical

products are used for preparation of the culture media and the

diluent.

13

384

NF' T 72-170

7.2.17.2.1.1

Gelose for counting

compos i t ion

q.s.p. water

2. ') 9

5.0 q

1. 0 g

12 to 18 g depending on

the ge1ifying properties

of the product

1000 m1

Dehydrateò yeast extract

Casein tryptic peptone

G i ucose

powdered agar-agar

7.2.1.2 Preparation

Dissolve the constituents or the complete dehydrated medium in

boiling water. If necessary adjust the pH so that aftero. 0scerilization it is 7.2 + 0.2 at 20 C (measured at 45 C with

temperature correction).

Divide the medium between test tubes at 15 ml per tube or between

flasks with capacity not greater than 500 ml at approximately

half the volume of the flasks.

Sterilize in an autoclave at 12loC + loC for 20 minutes

Before starting the test, place the gelose media in surfu~ion~ state

which have been previously cooled in a water bath at 40°C - 450C.

7.2.2 Gelose for preservation

7.2.2.1 Composition


soya peptone

Sodium chloride

of strains

powdered agar-agar

15.0 g

5.0 9

5.0 g

12 to 18 g according to thegelityi~g properties of theproduct1000 mlq.s.p water

14

385

NF T 72-1707.2.2.2 Preparation

Proceed as for the qelose Eor counting, but divide hetween tubes

(at 8 ml per tube) or stoppered Elasks oE suitable dimensions.

Sterilize in an autoclave. When they are removed Erom the

aut 0 cIa v e, k e e p the tu b e ~ ¡ nan inc lin e d po sit ion u n til the y arecompletel y cooled,

7.3 Bacteria suspension liquids

The liquids used are tryptone/sait solution Eors~~,s 6.1.1 to

6.1.4 and water (7.1) for strain 6.1.5.

7.3.1 Composition of the


Sodium chloride

Water

t ryptone/sal t solution1.0 g

8.5 g

1000 ml

7.3.2 Preparation

Dissolve the components in boiling water. Adjust the pH so that

after sterilization it is 7.2 + 0.2 at 20oC~

Distribute the liquid and sterilize in an autoclave at l2loC +

iOC for 20 minutes.

Just before prepari ng d i lut ions 0 f the bacter ial suspens ions,introduce the liquid into sterile test tubes, 9 ml + 0.1 ml of

liquid per tube.


The most suitable neutralizing agent for each product is selected

according to the results of the preliminary test.

It is prepared at double the concentration that should react in

the tests.

A list, which is not exhaustive, is given in Appendix C.

15

~~~

NF' T 72-170

The neutralizing ag~ni shall be sterile.

7.5 Specific interfering substances

7.5.1 Albumin/yeast extract mixture

a) Desiccated hovine albumin (Cohn fraction V for Dubos mcòium):

prepare a 3% solution (m/V), adjust to pH 6.8 - 7, if necessary,

using sodium hydroxide, sterilize by filtering over a membrane,

b) dehydrated yeast extract for bacteriology: prepare a 15%

solution (m/V), adjust to pH 7 with sodium hydroxide. Sterilizein an autoclave for 20 minutes at 121 0ç ~ 1 °C,

c) preparation of the mixture: aseptically mix 100 ml of albumin

solution and 20 ml of yeast extract solution. A solution with

2.5~ of each component is obtained. The final concentration in

the test will be 1% for the albumin and for the yea~i extract.

7.5.2 Hard water

2.21 gil solution of Caci2, 2 H20 in distilled water adjusted to

pH 6.5 - 6.8 by 1 or 2 drops of concentrated sodium hydroxide

solution, sterilized in an aULoclave for 20 minutes at 1210C +

lOC (the solution obtained is slightly opalescent).

The final hyrotiirtric ti~ in the test is 12 milliequivalents (60

French degrees).

7.5.3 pB 5 and pH 9 buffer solutions

a) Prepare the solution:

Sodium acetate, 3 H20 34.9 g

Sodium borate, 10 "20

~ipotassium phosphate

q.s.p. distilled water

95.3 9

43.5 9

1000 ml

b) Once the salts have dissolved, the pH is adjusted to the

required value using H2S04, 2.5 molll (for pH 5) or NaOH, 5 molll

(for pH 9).

16

Nf T 72-170

The two buffer solutions are sterilized by filLering or

autoclaving for 20 minutes at 121°C ~ laC, subJect to

verification of the r~ after sterilization.

7.5.4 Mixtures of albumin and hard water for clean and dirty

cond i t ionsThese mixtures enable test conditions to be created which arc

equivalent to those specified in the clean and dirty conditions

of the Counci 1 of Europe i s test methods for disinfectants in food

hygiene (Strashourg 1987). According to the conditions of use

specified for the product, one or other of the mixtures will be

used for the tests.

a) preparation of hard water concentrated 2.5 times

- Solution A

Anhydrous Mg Cl2 31.74 g

Anhydrous caCl2 73.99 g

Dissolve in 1 litre of distilled water and sterilize by

filtration or in the autoclave.

- Solution B

NaHC03 56.03 9

Dissolve in 1 litre of distilled water and sterilize by

filtration.

Put at least 50 ml of sterile distilled water into a 100 ml

sterile flask, add 0.75 ml of solution A and 1 ml of solution B.

Hake up to 100 ml with sterile distilled water.

The pH shall be between 7.6 and 8. The hardness expressed in

CaC03 is 750 mg/l, i. e. 75 French degrees.

17

388

Nf T 72-170

b) preparation of the albumin/hard water miXLure foe clean

condi tions

_ Dissolve 0.75 g of bovine albumin (Cohn fracLion V foe Dubos

medium) in ioa ml of hard water,

- Sterilize by membrane filtration.

With this mixture the final concentration in the test will be

0.3% for the bovine albumin and the hardness will be 30 French

degrees.

c) preparation of the albumin/hard water mixture for dirty

conditions_ Dissolve 2.5 9 of bovine albumin (Cohn fraction V for Dubos

medium) in 100 m1 of hard water,

- Sterilize by membrane filtration.

with this mixture the final concentration in the test will be l\

for the bovine albumin and the hardness will be 30 French

degrees.

8 APPARATUS AND GLASSWARE

8. 1 ApparatusS~andard laboratory equipment, in particular:

- Dry heat steril iz ing apparatus- Wet heat sterilizing apparatus

_ Bacter.ical incubator ventilated if possible and non-

dehyd~g, ensuring an even temperature of 37°C + 1 °C.

- Bath of boiling water

_ Water bath set to 40°C - 45°C

_ Water bath set to 20°C + i °C or 32°C + i °C

- Analytical balance

_ Stirrer that creates a vortex-type agitation (swirl)

18

389

NF T 72-170

- pH meter

- Spectrophotometer fittp.o with a monochromator set to waveiength

620 nm + 20 nm or photometer fitted with a filter centred on

this value

- Time r

8.2 Glassware and small equ ipment

- Standard laboratory equipment (tubes, 50 ml conical flasks,flasks with screwed stoppers)

- Petri dishes (preferably sterilized by radiation)

- 2 to 4 mm glass balls

- Spectrophotometry tanks wi th parallel sides having an optical

distance of 10 mm

To prevent problems related to recycling the glassware, the tubes

containing the product and its dilutions or the microbe

suspensions and the pipettes shall be made of disposable ster i leneutral glass.

8.3 SterilizationDry sterilization at a minimum of 1700C for at least i hour, or

1800C minimum for at least 30 minutes.

Humid sterilization at 12loC + lOC for a minimum of 20 minutes

- Sterilization by filtration over membranes with 0.45 pm pores.

9 PRELIMINARY TEST

9.1 Preparation of the bacterial suspensions

Note: the suspensions of S7æns 6.1.1 to 6.1.4 are prepared inthe liquid (7.3.1) while the suspensions of strain 6.1.5

are prepared in distilled water (7.1). These suspensions

shall be used within 2 hours of being prepared.

9.1.1 Strali6.l.1 to 6.1.4

Using a loop, take the str~6.l.1 to 6.1.4, cultivated as in

6.3, from the gelose medium, without breaking the surface of the

gelose.

19

390

NF T 72-170

srread the sample on the wall pf a sterile tube cont~ining

the liquid (7.3.1), the wall being humid, buL noL immer~ed.

Make a homogeneous suspension in the liquid phase by swirling

(for approximately 3 seconds).

~septically take a defined volume of this cuspension, adjust it~

absorbance Lo 62Q nm! 20 nm by diluting with the liquid (7.3.1)

and adjust with the spectrophotometer or photometer to obtain, in

the tanks with 10 mm optical distance, a value between 0.2 and

0.3 for $t"ilS 6.1.1 and 6.1.2 and 0.3 to 0.45 for s:r.:i~s 6.1.3 ann6.1.4.

The adjusted suspension shall contain between 1 x 108 and 3 x 108

cells per millilitre. It may only be stored at .: temperature

between ISOC and 220C for a maximum' of 2 hours.

9.1.2 Strain 6.1.5

Users' attention is drawn to the fact that great care is required

when handling this strain and that it is essential to observe

this operating procedure to ensure the qual ity of the results.

Wet the steri~ized glass balls in the conical flask, with a

maximum of 2~ or 3 drops of distil led water (7.1). using a loop,

take the culture from the solid medium and deposit the cells amid

the glass balls. Depending on the cellular density required, 5

or 6 samples may be taken. hgitate by rotating vigorously by

hand for 2 to 3 minutes. Add a volume of distilled water (7.1)

that is 4 or 5 ml greater than the volume required.

Agitate (2 to 3 rotations), rest for 10 minutes with the conical

flask inclined at 450.

Using a pipette, aseptically take the supernatant substance (in

one go) avoiding scratching the glass balls, transfer it to a

sterile tube and allow to decant for 10 minutes.

20

391

Nr T 72- i 7 0

Tak~ ufE the upper part and aseptically prepare th~ innciilumadjusting the absorbance with distilled water (7.1) as in 9.1~1

to a value between 0.4 and 0.5.

The adjust~d suspension shall contain between 1 x 108 and 3 x lU8

cells per millilitre. It may only be stored at a temperaturea 0between 15 C and 22 C for a maximum oE 2 hours.

9.1.3 Preparation of the test suspensions (preliminary test)

At the moment oE the test, dilute the suspensions adjusted as in

9.1 using the liquid (7.3.1) for str~~s 6.1.1 to 6.1.4 and

distilled water (7.1) for &ræi 6.1.5, changing pipette for each

dilution, so that they contain 1 x 103 to 3 x 103 cells per

mi11i1itre.

9.2 Bacteria count in suspension 9.1.3

Dilute 1 ml of suspension in 9 m1 of liquid 7.3.1 for s~~ins

6.1.1 to. 6.1.4 and in distilled water (7.1) for str~n 6.1.5.

Perform a control count taking, after agitation, two 1 ml samples

from each of the suspensions thus diluted and incorporating them

respectively in 15 ml - 20 ml of gelose for counting (7.2.1) inthe surfusion state at 400C - 4SoC. After solidification, turnthe dishes over and place them in the incubator (9.3.4).

9.3 Verification of neutralization in the presence of

interfering substances

- Define and note the concentration C of the product to be

tested: maximum bactericidal concentration specified in the

main test.

The operations described in Sections 9.3 to 9.5 shall be

performed for each strain.

21

392

NF T 72-170

9.3.1 Neutralization test

Distribute 4 ml of the inLer(ering substance (7.5) in a sieriletest tube, add 1 ml of waLer (7.11 and then, st~rtinq the timer,

add 5 ml of the product ~t concenLration 2C. Leave in contact inthe water bath for 5 minutes + 5 s, then transfer 1 ml of the

mixture to the test tube containing 4.5 ml of neutralizing agent

and 3.5 ml of water (7.1), which has been previously kept in the

water bath at 200C + 1°C or 32°C + 1°C. Leave in contact in thewater bath for 10 minutes + JO s.

carefully avoiding the walls of the tube, introduce 1 ml of the

bacterial suspension (9.1.3).

Start the timer when the addition begins and swirl for several

seconds. Leave in contact in the water bath at 20°C + 1°C or

32°C + ioC for 5 min + 5 s precisely.

At the end of the contact time very quickly swirl, take two 1 ml

samples of the mixture, transfer them respectively to Petri

dishes and incorporate them in 15 ml - 20 ml of the gelose medium

(7.2.1) in the surtusion state at 40°C - 450e. After

solidification turn the dishes over and immediately place them in

the incubator (9.3.4).

9.3.2 Interfering substance reference

Distribute 4 ml of the interfering substance (7.5) and 5 ml of

water (7. l) in a sterile test tube. Place in the water bath at

20°C + 1 °c or 32°C + 1 °c to establ ish thermal equilibrium.

Carefully avoiding the upper part of the tube walls, introduce 1

mi of bacterial suspension prepared as in 9.1.3. Start the timer

when the addition begins and swirl for several seconds. Leave in

contact in the water bath at 20°C + lOC or 32°C + .ioc for 5minutes ~ 5 s prec is ely .

22

393

NF'T72-l70At th~ end of the cont~ct time very quickly swirl, take two i ml

samples of the mixture, transfer them respectively to Petri

dishes ann incorporate them in 15 ml - 20 ml of gelose medium

(7.2.1) in the ~ur~ûsion state at 40°C - 45°C. After

. solidification, turn the dishes over and placp. them immediately

in the incubator.

9.3.3 Neutralizing agent reference

Distribute 4.5 ml of neutralizing agent (7.4) and 4.5 ml of water

(7.1) in a sterile test tube. Place in the water bath at 200C +

loC or 320C + loC to establish thermal equilibrium. Carefully

avoiding the upper part of the tube walls, introduce 1 ml of

bacterial suspension prepared as in 9.1.3. Start the timer when

the addition begins and swirl Leave incontact in the water bath for 10 min ~ 30 s at 200C or 320C. ~t

the end of the contact time very quiekly swirl, take two 1 ml

samples of the mixture, transfer them respectively to 2 Petri

dishes and incorporate them in 15 ml - 20 ml of gelose medium

(7.2.1) in the ~urÍusion state at 40°C - 45°C. ~fter

solidification, turn the dishes over and place them immediately

in the incubator (9.3.4).

9.3.4 Incubation

Incubate the dishes corresponding to counts 9.2 and 9.3, with thecovers underneath for:- 4 8 h r sat. 3 7 ° C + 1 ° C for stai s 6. 1. 1 to 6. 1 . 3

- 72 hrs at 37°C + 1°C for strain 6.1.4

- 7 days at 37°C + loC for strain 6.1.5.

(Take the necessary steps to avoid desiccation of the media).

9.4 Reading

9.4.1 Count the colonies that appear from' the dishes 9.3.1,9.3.2,9.3.3 for the neutralization test, the interferingsubstance reference and the neutralizing agent reference.Calculate the corresponding averages n', n'l, n'2 respectively.

23

394

NFT72-170

9.4.2 Count the colonies that appear from the rlishes 9.2 and

calculate the average N.

9.4.3 Note the neutralizing agents that, for each strain, aresuch that at least 50% of the bacterial cells are protp.cted from

thp. antibacterial action of the product, i.e.:n' ) n'2 or 0.5 n'22"

It should be ver ifien that the values of n' 1 and n i 2 are

equivalent to N, which signifies that the interfering substance

and the neutralizing agent have no effect on the bacterialsuspension.

9.5 validity of the bacterial suspension and selection of

neutralizing agent

9.5.1 verify that N as obtain~d in 9.4.2 is between 100 and 300.

9.5.2 Verify that n' ) 0.5 as shown in 9.4.3.

9.5.3 From the neutralizing agents tested, use the most active

and note the concentration C for which this result is obtained.

The neutralization conditions shall be considered as valid for

the tests when the concentrations of bactericide determined

during the main test do not exceed the concentration given above.

If necessary, different neutralizing agents, but ones which are

suitable for each strain, may be used for the main test.

9.6 Any exper iment plan may be used that respects the test

principles in 9.3.1 and allows the following to be obtained in

the neutralization test tube:

1) the product to be tested at.a concentration equal to

concentration C, the maximum concentration of bactericide

specified for the main test, and the interfering substance at the

final concentration laid down by the standard (see 7.5).

24

395

NF T 72-170

2) after 5 minutes' contact, the cìilution in the nelltra1i7.ingagent at the concentration that reacts ~ffectively in'thp. tp.sts,

3) addition of inoculum so that the final concentration in thp.cells in the test and the references is 1 to 3 x 102 cells per

millilitre.

10 MAIN TEST

Note the concentration C defined in the preliminary test.

Determinat ion of the bacte r icida 1 acti vi ty involves reproduci ngthe preliminary test (9.3) validating the neutralizing agent and

simultaneously performing the main test as indicated below.

The operations described in Sections 10.1 to 10.4 shall be

performed for each strain.

10.1 preparation of the suspension for the main testAt the moment of the test, prepare the bacterial suspension as

indicated in 9.1.1 and 9.1.2.

The adjusted suspension that has 1 x 108 to 3 x 108 cells per

millilitre serves as the test suspension. It may only be stored

at a temperature between lSoC and 220C for a maximum of 2 hours.

10.2 Bacteria count in the test suspension

10.2.1 Make dilutions of the test suspension to' 10-6

transferring 1 ml of the adjusted suspension prepared as in 10.1

¡ to 9 m1 of the liquid for preparing the bacterial suspensions

(7.3.1) for strains6.1.l to 6.1.4 and distilled water (7.1) forstrain 6.1.5.

Keep the 10-5 dilution to validate the neutralization method

(9.1.3).

25

NF T 72-170

10.2.2 After swirlin~, take two 1 ml samples, transfer each of

these to a Petri dish and then immediately incorrorate them in 15

ml - 20 ml of the gelose medium (7.2.1) in the surfusion state

at 40°C - 45°C. Aftp.r solidification, incubate the dishes as in10.3.4.

10.3 Testing the product

10.3.1 preparation of dilutions of the product

According to the presumed degree" of activity in the product under

test, for each strai~ prepare a range of dilutions of the product

in distilled water (7.1), with the product at twice the

concentration desired to be brought into contact with the

bacteria. By way of example, these dilutions may be selected

from the table below:

Table 1

Final concentrations in 'lm/v'or V/V

7.5 0.75 0.07550111 5 0.5 0.0525 2.5 0.25 0.02510 1 0.1 0.01

eic.

(7 J 'est concentrations greater than 50 , uy

not be ~tudied in accordance with this

standird. If this is necessary, the tests.ay be perfor.ed on a batch of antiseptic ~disinfectint that has been specially .anu-

fictured it twice the concentritionof. the co..ercial For.uhtion.

- .

Note: none of the concentrations shall be greater thanconcentration C defined at the beginning of Section 10.

Place the dilution in the water bath at 20° C t ioc or 32° C t 1° C

26

397

Nf T 72-170

10.3.2 Preparation of the neutralizing agent

In as many sterile tubes as there ñrp. dilutions of the product to

be tested, distribute 4.5 ml of neutralizing agent and then 4.5 ml

of distilled water (7.1). Place them in the water bath to

obtain thermal equilihrium.

10.3.3 Test

a) In as many sterile tubes as there are dilutions of the product

to be tested, distribute 4 ml of "the interfering substance.

Place in the water bath at ZOoC + 1°C or 3ZoC + loC to obtain

obtain thermal equilibrium.

b) Into each of these tubes, introduce 1 ml of the suspension

10.1 with 1 to 3 x 108 cells per millilitre carefully avoiding

the upper part of the tube walls. Start the timer at the

beginning of the addition and swirl for 3 to 5 s. Return to the

water bath at 200C + loC or 320C + 1°C for 5 minutes exactly.

c) At the end of the 5 minutes' contact, introduce into

these tubes, 5 ml of each dilution of the product 10.3.1. Start

a timer when the addition begins and swirl (3 to 5 s) at the end

of the addition. Place in the water bath for 5 minutes + 5 s

precisely.

d) At the end of the contact time very quickly swirl, take 1 ml

of the mixture and transfer it to the neutralizing agent prepared

as in 10.3.2. Replace in the water bath at 20°C ~ 1°C or 32°C ~

1°C. For this test to be valid, the timer shall not indicate atime greater than 5 minutes and 10 s at the end of the operation.

e) 10 minutes ~ 30 s after transfer to the neutralizing agent,

take two 1 ml samples of the mixture to count the number

surviving. Procede as in 10.2.2.

27

398

NF T 72-170

f) At the end of the test measure the pH tn the tubes where the

bacteria have been in contact for 5 minutes with the product, at

the maximum and minimum concentrations of the product to be

tested.

10.3.4 Incubation

Incubate the dishes corresponding to the counts before contacL

with the product 10.2.2 and after cnntact with the product

10.3.3, with the lid on, for a period of:

- 48 hrs at 370C + loC for $train~ 6.1.1 to 6.1.3-- 72 hrs at 370C + 10C for s~rai n 6.1.4- 7 days at 370C + 10C for str a in 6.1.5.

10.4 Reading

10.4.1 Group the dishes corresponding to the counts in the

preliminary test (9.3) and those of the main test (10.3.3).

10.4.2 Count the colonies obtained in the dishes 9.3.1, 9.3.2,9.3.3. Calculate the corresponding averages, n', nIl, n'2

respecti vely.

10.4.3 Count the colonies obtained in 10.2.2 and calculate the

correspondi ng average N.

10.4.4 Count the colonies obtained in 10.3.3, calculate theaverage n, the average of the 2 tests performed for each dilution

of the product.

11 INTERPRETATION

11.1 Verify that N obtained in 10.4.3 is between 100 and 300

colonies, which corresponds to a bacterial suspension with

1 to 3 x 108 cells per millilitre.

11.2 Verify that n' ~ 0.5 n'2, and that the values of n'l and

n'2 are equivalent to N.

28

399

NfT72-170

11.3 If the conditions defined in 11.1 ann 11.2 are s~tisfied,

for each sLrain of bacteria, finn the concentrations of the

product for which:

n ( n'2, which corresponds to a reduction of 105 times the number-~of bacterial cells.

11.4 If the conditions defined in 11.1 and 11.2 are not

satisfied, the test shall be rejected.

12 EXPRESSION OF RESULTS

12.1- products which are considered as spectrum 5 bactericides in the

presence of the interfering substance tested are products for

which the conditions required in 11 are satisfied for strùiòs6.1.1 to 6.1.5,

- products which are considered as spectrum 4 bactericides in the

presence of the interfering substance tested are products for

which the conditions required in 11 are satisfied for strains6.1.1 to 6.1.4.

12.2In the absence of other application standards, the following may

be proposed as concentrations for application:

-spectrum 5 in the presence of the interfering substance tested,

the minimum concentration of the product satisfying the

conditions required in 11 for straUs 6.1.1 to 6.1.5.

-spectrum 4 in the presence of the interfering substance tested,

the minimum concentration of the product satisfying the

conditions required in 11 for mx~ 6.1.1 to 6.1.4.

29

~o

NfT72-l70

13 TEST REPORT

The test report shall refer to this standard and specify:

- spect rum 4 0 r spect rum 5,

- the type of interfering substance.

It shall indicate:

- all details necessary for identification of the test (name,

manufacturer, batch no., nature and concentration of the active

substances) and specify the type of. diluent (generallydistilled water).

It shall mention the experiment conditions of the test:

- test temperature 200C or 320C,

- origin of the st.ai (CIP - ATCC) and where necessary the

conditions for preserving them with reference, for example, to

the tentative standard, T 72-140,

- the stability of the mixture of product and interfering

substance during the test,

the pH in the tubes where the product was tested at the minimum

and maximum concentrat ion,

- any procedural details not specified by the ~tandard and any

incident likely to have caused a modification in the procedure,

- where applicable, the nature of the active substances.

For each 5train the results shall refer to the preliminary test,

stating the neutralization agent used.

30

401

Nf T 72-170

APPENDIX A

formin~ an integral parl of t~e st~ndarrl

EXAMPLE OF PRESENTATION AND WORDI NG OF A TEST REPORT

ON THE DETERMINATION OF SPECTRUM 5 BACTERICIDAL ACTIVITY

IN THE PRESENCE OF AN INTERFERING SUBSTANCE

(dilution-neutra1iz3tion method)

A. 1 LABORATORY WHERE THE TEST WAS PERFORMED

A.2 FULL IDENTIFICATION OF THE SAMPLE

Name of the product:

Batch No.:Manufacturer:Date received at the laboratory:Storage conditions:Date of analysis:

Active substance(s) and concentration(s):(optional)Appearance of the product and its dilutions:

z

Y

Manufacturer 's

Not specified

Clear, colourless

product, so 1 uble

in water

A.3 EXPERIMENT CONDITIONS

A. 3.1 Test temperature:

A.3.2 Diluent for the product

recommended by the manufacturer:

used in the tests:

20° C

watersterile distilled water

A. 3.3 Interfering substance

Type: 1\ albumin plus 1\ yeast extract.Stability in the presence of the product: no precipitate for 2 hrs.

A.3.4 Conditions for preserving the strahCsStraiïs preserved and controlled in accordance wi th T 72-140.

31

Æn?

NF T 72-170

A.4 NEUTHALIZING AGENT

A.4.1 Nature and concentration lecithin J i (m/V)

~,4.2 Method of prepùration andsterilization dissolution when hoL

and sterillZùtion inan autoclave

~.5 PRELIM1NARY TEST

A.5.1 Concentration "C" 5 i for all strains

A.5.2 Resu 1 ts of the exper iment

Concentration of the

I

Strains r.o1lection of origin Jndn'No. in the collection N n" n'2produc t .C. 19.4.2) (941) (941) (9.4.11

5 % (V/VI Psl!udomonas al!ruginosii 160 95 161 158CIPA 22

5 % lV/VI E schl!richiii coli 258 187 246 265CIP54127

5 % lV/V) StaphylocoCCus iJurl!us 110 72 102 112CIP 53154

5%IV/VI Enterococcus Ilil!cium 154 161 141 163CIP 58 65

5 % lV/VI Mycobactiirium sml!gmiiris 109 87 79 101CIP 7 326

N : inoculu. control; n' . neutralizing test : n" : interfering i.stNe referene : n'2 neutral i iinq aqct referene

32

403

NF T 72-170

A.5,) Vdlidlty of the neutcùl1zingagent (~,6,2)

Pseudomonas aecuginosaEscherichia coliStaphylococcus aureusEnterococcus faecium :Mycobacterium smegmatis

lonce n t C a tlCi13 notexceeding lie"

5 i (V/V)5 i (v/V)5 i (v/v)5 i (v/V)5 i (V/V)

A. 6 MA I N TEST

A.G.l Results of the experiment

I

N n n'1 n'2!

Strains (1043) (10421 (104.2) (10.':.2)n (10.4.4) pH

collectinn ur

I

origin and No. value Conceniration in pcrccntaQe Cone. Cnne.

Iin the collection between V Iv in contact with bacteria min max

I

1 00 ~nd lX

I0.5 0.75 1 2.5 5 0.5 % 5°'"

i

I Pseudomontls220 207 202 203 + + + 7 a 5.1 5.7

ItleruginoSlCIP A 22

\ E scl,,,ricfiitl co/i

210 199 196 201 + 120 0 a 0 5.3 5.9

CIP S4 127i 230 205 199 218; Siaphylococcus aureus + 0 0 a 0 5.5 5.9

¡ CIP 53 154 -i

¡Enterococcus Itlecium 290 241 218 263 .. .. .. .. 0 5,4 5,7

,CIP S 855Mycobacieflum 190 178 186 190 .. + T 0+ S 5.1 5.8

smegm.ii:sCIP 7 326.

+ = .ore than 300 colonies

N . inoculu. control . n' . neutralizing agent test ; n'l : interfering substance ref~e ;

n'2 : neutralizing agent reference : n : test. (N, n'l ..nd n' 2 are equi vlIl enl)

Are bactericides, the concentration for whichn'2ni;-10

33

404

NF T 72-170

A.6.2 Bactericidal activity of the product

At 200C product Z has a spectrum 5 bactericidal activity in the

pr~sence of albumin and yeast extract in accordancp. with NF T 72-

170. (1988).

In the absence of other application standards, and for uses

involving contact times and temperatures close to 5 minutes at

200C, the concentration 5i may be proposed as a concentration for

spect rum 5 use in the presence 0 f pr ote i ns.

34

~5

NF T 72-170

APPENDIX B

forming an integral part of the stñndacd

EXAMPLE OF PRESENTATION AND WORDING OP A TEST REPORT

ON THE DETERMINATION OF SPECTRUM 4 BACTERICIDAL ACTIVITY

IN THE PRESENCE OF AN INTERFERING SUBSTANCE

(dilution-neutralization mp.thod)

B.l LABORATORY WHERE THE TEST WAS PERFORMED

B.2 FULL IDENTIFICATION OF THE SAMPLE

Name of the product:

Batch No.:Manufacturer:Date received a t the laboratory:

Storage conditions:Date of analysis:Active substance(s) and concentration(s):

(optional)Appearance of the product and its dilutions:

B.3 EXPERIMENT CONDITIONS

B.3.1 Test temperature:

B.3.2 Diluent for the product

recommended by the manu facturer:

used in the tests:

20° C

z

Y

Manufacturer's

Not specified

Clear, colourless

product, soluble

in water

watersterile distilled water

B.3.3 Interfering substance

Type: 1\ albumin plus 1 i yeast extract.Stability in the presence of the product: no precipitate for 2 hrs.

8.3.4 Conditions for preserving the strains

Str~ns preserved and controlled in accordance with T 72-140.

35

406

NPT 72-170

B.4 NEUTRALIZING AGENT

A. 4.1 Nature and concentration lecithin Ji (M/V)

A.4.2 Method of preparation and

steel i lizationdissolution when haL and

sterilization in an

autoclave

6.5 PRELIMINARY TEST

6.5.1 Concentration. C. : 5\ for all ~trc\ins

6.5.2 Results of the experiment

Cnnr.entration

I Slrain~ collection of origin

and N

\

n'1I n'2n

I-C. Ho. in the collection (942) 1941\ 19411 19411 i

5 %(V/VI Pseudomonlls aeruglnosa 160 95 I 161 I 158 I

I i

i

CIP A 22i5 % IV/VI Escher/chiii coil 258 187 246 265

CIP 54127 ¡

5 % lV/V) Stiiphylococcus aureus 110 72 102 112 i

CIP 53154 !,

5 % iv/V) Enterococcus fiiec/um 154 161 141 163ICIP sass

N i noculu. control: n neutralizing test n'l intertenng :;bltance refe~e: n '2 : ncuualizinq act refere I

36

407

NF T 72-170

B.S.3 Validity of the neutralizingagent (9.6.2)

concenlròtions not

exccecling .C.

Pseudomonas aerug i nosa:Escherichia coli:Staphylococcus aureus:Enterococcus Eaecium:

5% (v/V)5% (v/V)5% (v/V)S i (V /v)

B.6 MAIN TEST

B.6.1 Results of the experiment

N n' n" n'2(1043) (10.4.2) (10.4.21 (104.21

n(1044\ pH

Str¡in"collection of

V.1Íi~ Concentration in pcreenlaqc

origin and No. in v/v in contact wi thCone. Cone.

between theco 11 ec t i on

m,n m3X100 and 300 b~caria

0.5 0.75 1I

2.5 5 0.5% 5 'C

Psiiudomonu 220 207 202 203 + + + 7 a 5.1 5.7¡iiruginosiiCIP A. 22

Escherichiii coli 210 199 196 201 + 120 a a 0 5.3 5.9CIP 54 127

St.phylococcus iiuriius 230 205 199 218 + 0 0 0 0 5.5 5.9CIP 53 154Entiirococcus /iiiicium 290 241 218 263 + + + f a 5.4 5.7CIP 5 855

.. = .ore th¡n 300 colonie,

N : inoculu. control n .neutrali zing agent test : n'l : interfering 'ub'tanr.e reference ;

n'2 : neutralizing aqent reference. : n : test (N, n'l and n'2 are equiwalent)

n'2Are bactericides, the concentrations for which n~--

10

37

408

NF T 72-170

B.6.2 Bactericidal activity of the product

At 200C product Z has a spectrum 4 bactericidal activity in the

presence of albumin and yeast extract in accnrnance with NF T 72-

170. (1988).

In the absence of other application standards, ann for uses

involving contact times and temperatures close to S minutes at

20°C, the concentration 5% may be proposed as a concentration for

spectrum 4 use in the presence of proteins.

38

409

NF'T 72-170

APPENDIX C

not an integral part of th~ standard

LIST OF SUBSTANCES THAT NEUTRALIZE ANTIBACTERIAL ACTIVITY

(not exhaustiv~)

For phenyl nroducts and quaternary ammoniums

- Lre5h egg yolk diluted to 5% or 0.5% (V/V)







5% fresh egg yolk (V/V)3% polysorbate 80 (l) (V/V)

Ji polysorbate 80 (l) (V/V)

0.3\ or 3% leci thi n (m/V)

3% ethylene oxide condensate on

fatty alcohol (2) (m/V)

2% lecithin (m/V)

3\ polysorbate 80 (1) (V/V)

O. 4\ ~odium sulphate lauryl (m/V)

0.3\ lecithin (m/V)

3i polysorbate 80 (1) (V/V)

1% saponin (m/V)0.3i or 3i lecithin (m/V)

7\ ethylene oxide condensate on

fatty alcohol (2) (m/V)

2i leci thi n (m/V)

4i polysorbate 80 (1) (V/V)

(i) Known under the trade name : Tween 80, analytical grade,non hydrol i zed

(2) For example : Lubrol W or Ci r rasol ALN W

39

410

NF T 72-170

For alrlehydes

- ?rp.p~r~tion contai~ing J\ polysorbate 80 (1) (v/V)o . J i 0 r 3 i 1 e cit hi n (m/ V )

0.1 \ L-histidine (m/v)

- preparation containing Ji polysorbate 80 (1) (v/V)

D.3\ or Ji lecithin (m/v)

0.1\ glycocoll (m/V)

For organo-mercurial products and products containing metals

- sodium thioglycolate at 0.05\ or 0.5\ (m/V)

- L-cystein at 0.08\ or 0.15\ (m/v)

- thiomalic acid at 0.075\ (V/V) brough to pH 7 by sodium

hydroxide

For halogenated der i vatives (hypochlori te, chloramine T,iodophors, etc.)- sodium thiosulphate at O.st (m/V)

For peroxides

- catalase

- peroxidase

For these two enzymes, one unit catalyses the

decomposition of 1.4 mole of hydrogen peroxide

per minute at 250C at pH 7

(l) Known under the trade namenon-hydrol i zed.

Tween BO, anal~ical grade,

40

411

Produc t

o be tested 2C

51

E

Sl

E

Ni

E

S ml--4 ml-1 ml--

4ml--Sml

4.5mf-4.5ml

..

Bdrleri~1~u~~ension

1 à 3.108 cell

8

.. 8

8

- 4 1 -

NF T 72-170

Diluent DouE (f\~0

~;./Dilutions-----~ 1 ml..._-.._-~(1 + 9l(V/VI

3103 cell

NI4.S ml

,; , JS mil ~I +i . +i:FmI8¡~u 1-- 1 I· i I UN 0M I IN~ I I ~I i 0 I~ I

oN

8uooI'

'"..L-

I ~II ..II~ :"1

IN :1I~ ::11° 1IV::'i. ::.0._1:N~ii -:1

o..

~8

, .,11 ~I:ct .:1iN "i1M ~iI~ :iiv::'I~ .,'IN "iI :. iI ._,.D....

'"

8o..

c = "aii.u. bactericidal concentration specifiedNt = Double concentration neutralizing ag'en t

Sl = Interferi ng substance0 = Diluent 1.3

E = "ater 7.1

~~"6'" 1 00 to 300 coloniesin

ß/O+i iC

.eII

I

v

'~ö.I'ML-aV.0I'

IIIt

i +i 'i C Ii .e I1 It :i I iI

I ~ II I' 1

1 M II ~ ii v :.oN

0..'.. ......\ . .. ..~.......

~ö..o

1MI T1

I.SI Ei ~i I

i vi 0iN1MI .~

VooN

CD.....::.

. . ":.. .\.:.,CC~ . ....:..)/ :,..:.:'

--ft/9.3.3 .n'2

~o.. .. :'..

Figur.' -- Diagra. of preli.inary test for a bacterial strain, a neutralizing agent and

an interfering substanee

412

Product

to br i~sird

S I

E

51

c =..1 =

51 =

o =E =

- 42.. NFT72.170

EE

5 ml'-8 u

o

lml g:-- ii

I

i

.. ..

?"I

uo iNM N

M4 ml-- , ii iU

, 0

; 0N

L.o1 ml--

Ni 4.5;lE 3.5 ml

'0N

E

--.--.--:----.--1' :.êi '-'-4ml gd _E!lgi~:~O-- i ~I i N I5 ml ii' ..I I 11' I ~ n',-- :~~: ! / ¡ ~: i"'~o,,: ..i~ ~r I i ~ I :..o ~ ,

--"--"~~-.--_.-_._L "_ ._:.~: i E .i: .;1 1- i E r . .. ;.,4.Sml g! ~- I'"u I4.5 ml 10 ::,- rc::1I.. :;1 1 u 1 .' . . .,..1° .., ~ ~ I. ..i' :: .. ~ I ~ ..' . .'. ....00 ..' .:N~ ", .-- --- - .. .. --

Dilutions 11 + 911VIVI' ..: ~ ,· , 9 ml 0 or E . ::. . .. :..::Producllo bl! tuhd it rl/ :. ' '....'~

, "'ë'OIoo' , t. '.l"'~1I ~C2c., ., - 4,5 mii ..., -..-Ë ~ .' 4.6 ml i E i :' .';. ....~;:.. E - i 0 I ." '.

4j~ ~N~:.I 5 mg,! ~¡ : ~ : n~~ : 1 ml_ : ~ : ~ .'. .,:::.:.~)..~ i I ~ i ::' ::;'::'.:~'b i I · i .," ..' .......~' I ~ i ....:..oN

51

Nt

E

1I1tl!r bath

Kaii.u. bactericidal con~entrationspecifil!d

Double concentration neutralizing igent

lnterfering substance

Di luent 7.3

lIater 7.1

Figure2- Actual test and simultaneous repetltion of the

preliminary test for a neutralizing agent, a

bacterial strain and an interfering substance

(

(c A Clinica and Experimental Dermatology (1980) 5, 197-201.

A comparion of the antimicrobial effect of 0.5%chlorhexidie (Hbistat~) and 70% isopropyl alcohol

on hands contaminated with Serratia marcescens

(

RAZA ALY*t AND HOWARD I.MAIBACH** Departmet of Dertology and t Departmet of Microbiology, University of California, SanFrancsco, CA 94143, U.S.A.

Accepted for pulication IS August 1979

(

(

SumarTwo anticrobial preparations for handwashig were compared by the 'gloved hadwashmethod' in seventy-two subjects. The test preparation was 0'5% chorhexidie gluconatealcoholic-eollent hadwash (Hibistat'); the reference agent was 70% isopropyl alcohoL. The

hads were contaated with Seratia marcesce and disineced with each antieptic twenty-five ties over an 8 h day. Bacterial counts were obtaied for each hand after intial contaa-tion, and after 5, 10, IS, 20 and 25 contaation/treatment procedures.

There was a statistically signcat reducton in recovery of S. marcescen after ch0rhexidietreatment compared to alcohol (P.: 0'01). When recovery of organms was plotted agt thenumber of handwashes, there was a signcat liear reducton in trient Bora for chlor-

. hexidie treated hands (P.: 0'01) but not for alcohol treated hands (P.: 0'20).(

( \Cutaeous bactera are conventionay classifed as 'trients' and 'residents' (price, 1938).The trients are those present on the ski and acquied mostly from envionmenta contact.

They mayor may not temporary colonie the ski and may be removed easily by a simplewash procdur. To qualiy as residents, organsms must multiply on the ski and not merelysurve. Under normal conditions, these micro-rganms remai faily constant.

Ths investigation compares the effcacy of ch0rhexidie gluconate alcoholic emollenthadwash (Hbistat*) and 70 % isopropyl alcohol agait the contaatig organsms (trient

Bora) on the hands. The study simulates hand washig by heath cae personnel over the coureof an 8 h day.

(

0307-6938/80/060197 So2.CO (C1980 Blackwell Scientific Publications 197

( F

(

Antîmîcrobia qJect of Hîbîstai- 199

Pigment producig Seratîa marcesce maitaed in our laboratory were adjusted toconta 108 colony form unts/mI.

Test preparations were chlorhexidie gluconate alcoholic emollent handwash and 70%isopropyl alcohoL.

Results

Quatîtatîiie recO' of artifcîa1ly appHed blUtena

The bacteri recovery from the contaated hads durg the baslie period was compared

to test the simarty of the two populations (chlorhexidie vs. alcohol groups). The logio

Table J. Anticrobial efec of chorhexdie gluconate alcoholic emollent and 70%

ispropyl alcohol agait the contamatig bacter

Mea logio number of bacter recvered frm the hads

Numbe of washes

ChorhaidietAlcoholt

Baselie.6'2322

S'9409

52'7829

4'2656

101'49804'3385

IS1'0788

4'1254

200'6729

3'8763

251"0264'oS68

· Hands were contated with Seratia. mauscm, but not wahed with the testpreparations.

t Th-six subjecs were used for each test prepartion.

6.

14* 3

oJ

e-e_e_e_e

2 \~ ---..---5 io 15 20 25

Baseline Number of washes

Fig J. Bacteral counts of hads before (baselie) and afer treatments with chorhexdie

gJuconate C..-..) or 70% isopropyl alcohol C.-e) at Sth, ioth, ISth, 20th, and 25thwashes,

Antimicrobial effect of Hibistat. 201

known ski disinectant and accounts for most of the rapid anticrobial effect (Byan &Henderson, 1973). An antiseptic must not only achieve an imediate substantial reducton of

the microbial Bora, but also retai its anticrobial actvity to provide protecton agait the

next contamation of the ski. In ths study, five logs and more than one log reducton (cacu-

lated from the baselie) were noted after twenty-five contamatig/washig procedures withchlorhexidie and 70% alcohol respectvely, thus suggestig that chlorhexidie has muchgreater potent bacteriocidal actvity agaist Seratia marcesen than alcohoL. Alcohol alone wasincapable of maitaig a substantive antiseptic effect on the ski; it has presumably lostmuch of its anticrobial effect on evaporation.

Acknowledgments

Dr John Sted! provided statistical assistance and Charlene Litz and Joan Colman techncaassistance.

References

HYATT, M.E. & HENERSON, A. (1973) Preoperative sterzation of the perieum: a comparison of six

antiseptics. Journal of Clinical Pathology, 26, 921"'24.DINE, P. (1978) Handwashig degermg: a comparison of povidone-iode and chlorhexidie. Clinical

Pharmacology and Therapeutics, 23, 63-67.FEDER REGISTER (FDA) (1974) Volwne 39,179: 33136.PETEON, A.F., ROSENBERG, A. & AiATARY, S.D. (1978) Compartive evaluation of surgical scrb

preparations. Surgery, Gynecology and Obstetrics, 146, 63-65.PRICE, P.B. (1938) The bacteriology of normal ski: a new quantitative test applied to a study of the

bacterial flora and the disinectant action of mechanical cleansing. Journal of Infectious Diseases, 63.301-318.

RUSSELL, A.D., MORRIS, A. & AiLWOOD, M.e. (1973) Methods for assessing dage to bactera induced

by chemical and physical agents. In: Methods in Microbiology (Ed. by J,R, Norrs and O.W.Ribbons), VoL. 8, pp. 95-182. AcademicPress, New York. '

(

ll c:'CÚct.vi _()~ (.j .rv'

.IED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1989, p. 3113-3118

.2240189/1231 B-06$02.0010:right ~ 1989, American Society for Microbiology

Vol. 55, No. 12

In Vivo Protocol for Testing Effcacy of Hand-Washing Agentsagainst Viruses and Bacteria: Experiments with

Rotavirus and Escherichia coli--_.~-SHAMIM A. ANSARI,! SYED i. SATIAR.Ì~ V. SUSAN SPRINGTHORPE,! GEORGE A. WELLS,2 AND'~ERTOSTOWARYK2

'epartment of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, CanadaKI H 8M5,! and Division of Biometrics, Laboratory Centre for Disease Control, Health and Welfare Canada,

Ottawa, Ontario, Canada KIA 0L22

Received 13 June 1989/Accepted 8 September 1989

Ten antiseptic formulations, an unmedicated liquid soap, and tap water alone were compared for theircapacities to eliminate human rotavirus from the finger pads of adult volunteers; three of the antiseptics. thesoap, and the tap water alone were also tested against Escherichia coli. A fecal suspension of virus or bacteriumwas placed on each finger pad and air dried. The contaminated site was exposed to the test product for 10 s,rinsed in tap water, and dried on a paper towel. The residual virus or bacterium was then eluted. Selected

agents were also tested by an analogous whole-hand method by which the entire palm surfaces of both handswere contaminated. Alcohols (70%) alone or with Savlon reduced the virus titer by ::99%. whereas the

reductions by Proviodine. Dettol, and Hibisol ranged from 95 to 97%. Aqueous solutions of chlorhexidinegluconate were significantly less effective for virus removal or inactivation than 70% alcohol solutions.Furthermore, Savlon in water (1:200) was found to be much less effective in eliminating the virus (80.6%) thanthe bacterium (98;9%). The tap water alone and the soap reduced the virus titers by 83.6 and 72,5% and thebacterial titers by 90 and 68.7%, respectively. The results of the whole-hand method agreed well with those ofthe finger pad protocol. We conclude that the finger pad method is a suitable model for testing the in vivoeffcacy of hand-washing agents and emphasize the need for using appropriate test viruses and bacteria.

ands are among the principal vehicles for transfer of;tious agents in ihe community in general and in institu-:11 settings in particular (4, 23, 25, 32). Regular andier hand washing is, therefore, considered an importantin preventing the spread of infectious diseases (15, 20),guidelines for this purpose are available for health careKer~ (12). The continued occurrence of institutional

"S of many enteric infeciions, particularly those,ed by rotaviruses (11, 20), is, however, believed to beto the lack of compliance with hand-washing guidelineser than the inadequacy of the hand-washing agents (16).arlier studies using the suspension test have demon-ted that rota viruses are not readily inactivated by many,s of commonly used chemical disinfectants (30); theseses proved 10 be even more resistant to disinfectionn they were dried on nonporous inanimate surfaces (18).:e products found to be ineffective in these in vitro:ies included certain commonly used antiseptics, we,idered it important to examine them and other similarjucts for their abilities to eliminate rota

viruses fromian hands.yen though the testing of hand-washing agents against

ses on experimentally contaminated human hands has:i studied before (8, 13, 14, 28), at present there is noidardized protocol available for such testing. So far, ini testing of such products has been conducted almostiusively with bacteria (3, 7, 19, 24). This study was,.efore, undertaken to design and test a simple in vivo¡ocol using finger pads for testing the rotavirus-elim-ing effciency of hand-washing agents. The protocol wasi applied to compare the rota virus-eliminating effcien-

Corresponding author.

cies of the hand-washing agents with their abilities to removeor inactivate Escherichia coli. Finally, experiments wereconducted with selected hand-washing agents to demon-

strate that the results with the finger pad protocol are

representative of those obtained when the whole hand isexperimentally contaminated with the virus or the bacteriumand washed in the normal manner.

MATERIALS AND METHODS

Cells. The MA-I04 line of rhesus monkey kidney cells wasused for the growth and quantitation of the rotavirus. Themethods used for the cultivation, mai,ntenance, and passageof these cells have been described in detail elsewhere (26).For the virus plaque assay, cell mono layers were prepared in12-well plastic plates (Costar, Cambridge, Mass.) with Eagleminimal essential medium (Flow Laboratories, Inc..McLean, Va.) containing 5% fetal bovine serum (GIBCOLaboratories. Grand Island, N.Y.) and gentamicin (Cidomy-cin; Roussel, Montreal, Quebec, Canada) at a final concen-tration of 50 ¡ig/ml. The seeded plates were sealed in plasticbags (Philips, Toronto, Ontario, Canada) and incubated at37°C for 48 h for monolayer formation.

Fecal sample. A sample of formed feces was obiained froma healthy l-year-old child. A 10% (wt/vol) suspension of thesample was prepared in normal saline. The suspension wascentrifuged at 1,000 x g for 15 min to remove coarseparticulate matter, and the supernatant was collected forstorage at ~20°C. It was found to be nontoxic to MA-I04cells, noninhibitory to the rotavirus, and free from anyagents that could produce cytopathic effects or plaques inthe cell line (2).

Virus. A cell culture-adapted strain (Wa) of human rota-virus obtained from R. G. Wyatt (National Institutes of

3114 ANSARI ET AL. AppL. ENVIRON. MICROBIOl.

Active ingredicrit(s) % Concn

TABLE 1. Hand-washing agents

Dilution Product (manufacturer)

isopropanolEthanolChlorhexidine gluconate and

cetrimideChlorhexidine gluconaie and

cetrimideChlorhexidine gluconate and

cetrimideChlorhexidine gluconate and

isopropanolChlorhexidine gluconale and

isopropanolPovidone.iodine

70.0 (vol/vol)70.0 (vol/vol)1.5 (wi/vol) and 15.0 (wt/vol)

1.5 (wt/vol) and 15.0 (wtlvol)

1.5 (wt/vol) and 15.0 (wt/vol)

0.5 (wt/vol) and 70.0 (wtlvol)

2.0 (wt/vol) and 4.0 (wt/vol)

10.0 (wt/vol) 0% free 12)

PCMX 4.8 (wi/vol) and 9.4 (vol/vol)

Liquid soap 100

Tap water

a NA. Not applicable.

Health. Bethesda. Md.) was used in this study. The proce-dures for the preparation of the virus pools and plaque assayhave been described earlier (2). For contamination of thefinger pads or hands, the virus suspension was prepared bydiluting it 10-fold in the fecal sample.

Bacterium. The strain of E. coli selected for this study wasisolated from the fecal sample used for suspending the

rotavirus. For the initial isolation and quantitation of E. coli,mFC agar (Difco Laboratories. Detroit. Mich.) was used.The test suspension of the bacterium was prepared by

mixing an 18-h broth culture with the fecal sample at a 1: 10ratio.

Volunteers. Permission of the Ethics Committee of theUniversity of Ottawa was obtained for the use of humanvolunteers; one adult male and two adult females partici-pated in this study.

Hand-washing agents. Ten antiseptic formulations, an un-medicated. liquid soap, and tap water were tested for theireffcacy in removing the rotavirus from experimentally con-taminated finger pads. Details of the hand-washing agents.tested are given in Table 1. Isopropanol (70%), Savlon in70% ethanol (1:30), Savlon in tap water (1:200). liquid soap,and tap water alone were also tested against E. coli.

Paper towels. Rolls of ordinary unbleached paper towels(Boudreau It, Inc.. Hull. Quebec, Canada) were purchasedlocally and used for drying treated finger pads or hands asspecified in the protocols.

Finger pad protocol. A modification of the protocol devel-oped earlier in our laboratory to siudy the survival ofrota viruses on human hands was used (2). The details of thisprocedure are given in Fig. 1, but only the results for themethod which included paper towel drying are reportedhere. The same basic procedure was also used with thebacterium, but E. coli was quantitated as CFU on mFC agarplates. Elution fluid for both the rotavirus and E. coli wasEarle balanced salt solution containing 20% (vol/vol) tryp-tose phosphate broth, and quantitation of the organisms waspenormed immediately after elution. At the end of eachexperiment, the contaminated finger pads of the volunteerswere thoroughly rinsed with 70% ethanol and then washedwith soap and water.

Whole-hand protocol. The following whole-hand washingprotocol was used to ascertain that the results obtained with

NoneNone1:30 in 70% isopropanol

NAnNAnSavlon (Ayerst. Montreal. Quebec:,

Canada)Savlon1:30 in 70% ethanol

1:30 and 1:200 in tap water Savlon

None Hibisol (Ayerst)

None Cida-Stal (Huntington, Bramalea,

Ontario, Canada)Proviodine (Rougier, Montreal,

Quebec. Canada)Dettol (Reckill & Colman.

Lachine, Quebec, Canada)Ivory (Procter & Gamble,Toronto, Ontario, Canada)

None

None

1: 10 in tap waler

our finger pad protocol were representative of the normal

hand-washing situation. The volunteers did not wash ordecontaminate their hands before the slaTt of the experi-ment. A 0.5-ml portion of the fecal suspension of the virus orthe bacterium was placed on the palm sunace of one hand.The volunteer was then required to spread the inoculum overthe entire palm sunaces of both hands by gently rubbing

them together. and the inoculum was allowed 10 dry for 20min under ambient conditions. To obtain the base titer of the

~-:~ ~~ 511"9dl 01 ~ ..nc ...1' ..eft inutled .11t!'OUL~'~'O~.,... 01"" Ilft;ill

inc~'.d '''ø-dl itiøoMØ to'mL 01 "',..." "0"11'" wI.,1

101 10 "con. _lt" 10 LUL """'WII~n~,Inie ",lin '5 mL lid .11'"'~;~~ D=_/. ~/ dried .11.. 10-'1

nold'"'' IØ-Ø.' N CIOlfi.

~-8 wt'd I'I.d wllh 1 mL -ultor '-0 MConl .111' 40 lul in"'eflioi

~~~FIG. 1. Procedure for in vivo testing of hand-washing agents.

11..55, 1989 HAND-WASHING AGENTS AGAINST VIRUSES AND BACTERIA

TABLE 4. Comparison of whole-hand and finger pad protocols..BLE 2. In vivo effcacy of hand-washing agents against human

rotavirus in linger pad protocol"

Hand-washing agoni(concn)

% Rediiciion(mean:! SDl

Tiikeygrouping

,propanol (70%).ianol (70%)vlon in 70% isopropanol (1:30)vlon in 70% ethanol (1 :30)iviodine1101

:iisol~uid soapvlon in water (1:30)

p waterla-Statvlon in water (1:200)

99.8 :! 0.18

99.8 :! 0.19

99.4 :! 0.84

99.1 :! 0.50

96.5 :! 2.65

96.2 :! 3.62

95.3 :! 2.15

86.9 :! 2.42

86.4 :! 4.07

83.6 :! 3.49

!l0.3 :! 3.51

78.3 :t 5.55

AAAAA, BA, B, CA, B. C0, B, C

0, Cooo

Agents with ihe same Tukey groupings do not have significanily differentc~.. .. .:~es of reduction (0 = 0.05).

;( organism present at the end of this drying period, 20 mlthe eluent (20% tryptose phosphaie broth in Earle bal.eed salt solution) was poured on the hands while they:re being rubbed together over a plastic funnel 27 cm inimeter in order to bring the eluent in contact with the

iire contaminated surface. Generally, more than 18 ml of~ eluate was collected by this procedure.To test the effcacy of a hand-washing agent, the hands

:re contaminated with ihe test organism and dried as

scribed above. Then 0.5 ml of the test agent was placed on~ palm surface of one hand. and the volunteer was requiredrub his or her hands for 10 s, Ihereby covering the entireniaminated surface. The hands were then washed by.uring over them 500 ml of tap water at 40°C and dried withpaper towel. The residual test organism was then eiuted1m the washed and dried hands, as described above. TheJates from the rota virus-contaminated hands (controls and,ts) were passed through separate columns of a Sephadex-1-20 gel (Pharmacia. Uppsala, Sweden) to remove anysidual cytotoxicity due to antiseptic chemicals (5). The

a , from the columns were then centrifuged at 10,000 x

,0 i cmove bacteria and fungi before the virus plaque assay.:uates from bacterium-contaminated hands were titrated¡mediately on mFC agar plaies.Statistical analyses. For the finger pad protocol, the testing. each hand-washing agent with each microorganism wasinducted at least three times by using two finger 'pads for~alment with the test agent in each triaL. Therefore, thesuits presented in Table 2 are ihe mean of six observationsr each hand-washing agent with one volunteer, and thoseven in Table 3 are based on the mean of six observations

Hand.washing ageni% Reduciion (mean:! SD)

and protocol" E. coli Roiavirus

70% IsopropanolWH ::99.9 :! 0.1 99.9 :! 0.1FP 99.0 :! 1. 99.8 :! 0.2

Savlon in 70% ethanol (1:30)WH ::99.9 :! 0.1 98.9 :! 0.6FP 98.7 :! 0.3 99.1 :! 0.5

Savlon in tap water (1:200)WH 96.6:! 2.2 84.8 :! 6.8FP 99.6 :! 0.1 78.3 :! 5.6

Liquid soap (1:10)WH 94.5 :! 3.2 93.8 :! 3.9FP 60.8 :! 36.8 86.9 :! 2.4

Tap waterWH 95.1 :! 4.4 85.5 :! 12.0

FP 90.0 :t 1.9 83.6:! 1.4

. Differences beiween whole.hand and finger pad protocols were nolsignificani for any agents iested.

b WH, Whole hand; FI', finger pad.

with three volunteers. For the whole-hand washing protocol(Table 4), .each experiment was repeated three times and thevolunieer used was the same as in experiments summarizedin Table 2.

Results were statistically analyzed by using two-wayanalysis of variance based on the arcsine transformation ofthe raw data, i.e., the percentage of organisms removed. Thearcsine transformation was utilized in order to stabilize thevariance. Comparisons of the means of results obtained withthe various hand-washing agents were made by the Tukey \Iprocedure (31). This procedure allows for multiple compar-isons of means while controlling for the error rate at thedesired leveL.

RESULTS

The challenge titers of the test organisms in the finger padexperiments were (1.5 :t 0.4) x 104 and (9.9 :t 1.6) x 104 forthe rotavirus and E. coli, respectively. These titers weremeasured at the end of a 20-min drying period. the minimumtime for a visually dry appearance of the inoculum underambient conditions, and they represent 57 and 7% of theoriginal inoculum for rotavirus and E. coli, respectively. Inthe whole-hand experiments, after 20 min of drying, (1.8 :t0.9) x 104 and (5.7 :t 3.2) x 104 were the challenge titers left

TABLE 3. Comparison of in vivo effcacy of hand-washing agents against human rotavirus and E. coli in finger pad protocol

3115

Volunteer 70% IsopropanolSavlon in 70% ethanol

(1:30)

Mean % reduction in infectivity tiier with:

Liquid soap Tap waier

Rotavirus E. coli Rotavirus E. coli

Savlon in water(1:200)

Rotavirus E. coli

99.899.699.5

99.099.799.2

99.198.899.4

98.799.199.0

90.088.191.8

Roiavirus E. coli

iverall mean 99.6:! 0.2 99.3:! 0.4 99.1:! 0.3 98.9:! 0.2 80.6:! 5.5 98.9:! 1. 72.5:! 5.1 68.7:! 10.5 83.6:! 1.4 9O.0:! 1.9:: SO

Rotavirus E. coli

77577387.0

99.697.699.5

78.171.468.1

60.880.664.7

83.682.284.9

I.... .

. .. )

l~a

~f.'~.-.í,...:~."

,3116 ANSARI ET AL.

on the hands. These amount to 22.9 :! 6.0 and 1.4 :! 1.3% ofthe original inoculum for the rotavirus and E. coli, respec-tively.

The results of testing hand-washing agents and tap wateragainst the rotavirus are shown in Table 2. Seventy percentisopropanol, 70% ethanol, and Savlon containing 70% ofeither one of the alcohols were each able to reduce the

infectivity titer of the virus by ~99.0%. Hibisol, a waterlessproduct containing chlorhexidine gluconaie and 70% isopro-

panol, eliminated 95.0% of the virus. Dellol ami Proviodineachieved 96.2 and 96.5% virus removal, respectively. Cida-Stat and Savlon in water (1:30 and 1:200) produced thelowest levels of virus removal of all the antiseptics tested, asthey were able to reduce the virus titer by only 80.3, 86.4,and 78.3%, respectively. Surprisingly, in terms of the meanpercentages of virus removal, the liquid soap (86.9%) and tapwater alone (83.6%) were as good as. if not beller than,Cida-Stat and Savlon in water (1:200).The percentages of virus removal by the various hand-

washing agents were statistically compared by using iheTukey II procedure. Results of the multiple comparisons

made by the II procedure of the means of the 12 hand-washingagents are displayed in Table 2. Those means designated bythe same leller under the Tukey grouping were considerednot significantly different from each other at the a = 0.05leveL. No jilJerences were seen among the hand-washingagents represented by the leller A. Likewise. no differenceswere demonstrated among those with the letter D. Group A,primarily the alcohol-containing formulations and those so-lutions with high concentrations of iodine or p-chloro-m.-

xylenol (PCMX), was significantly different from group D,which included the aqueous chlorhexidine salts, soap andwater, and water alone. However, multiple comparisons alsoshowed the existence of intermediate groups Band C, whichshowed some overlap with the means for hand-washingagents in groups A and D. Despite these overlaps, thealcohols or alcohol-containing formulations were clearlysuperior to water and the aqueous solutions of chlorhexidinesalts in reducing the levels of contaminating virus.

In a separate set of experiments using three volunteers,

four hand-washing agents and tap water alone were com-pared for their abilities to remove the rotavirus and E. colifrom experimentally contaminated finger pads (Table 3). Thedifferences in the mean percenl removals for the virus andthe bacterium were not statistically significani for the alco-hols, the alcoholic solution of Savlon, the soap, or the tapwater alone. However, there was a statistically significantdifference (P = -:0.0001) between the mean percent remov-als of E. coli (98.9%) and the rotavirus (80.6%) by Savlon inwater (1:200).

Statistical analyses of the data summarized in Table 3 alsoshowed that the person-to-person variation in mean percentreductions by a hand-washing agent against a given organismwas not significant (P = 0.14).

In order to determine that the finger pad protocol was

representative of the normal hand-washing practice. selectedhand-washing agents and tap water alone were also tested bythe whole-hand protocol for their abilities to remove the .twotest organisms. The results of these experiments are sum-marized in Table 4. Isopropanol (70%) and the alcohol

solution of Savlon reduced the titers of both the organismsby at least 98.0% in both the protocols tested. Even thoughthe overall removal of the virus and the bacterium by theother hand-washing agents and tap water alone was not ashigh as that seen with the alcohol solutions, there was nosignificant difference between the results obtained with the

ApPL. ENVIRON. MICROBIOL.

whole-hand washing and the finger pad protocols. The widevariations observed between the finger pad and whole-handexperiments with liquid soap and E. coli were not found tobe significant at the a = 0.05 leveL. For Savlon in water(1:200), the results with the whole-hand experiments weresimilar to those with the finger pad protocol; this solutionwas found to be significantly less effective in the eliminationof the virus (84.8%) than of the bacterium (96.6%).

DISCUSSION

Hand washing is regarded as the single most importantprocedure for preventing the transmission of infections (23).However, the impact of hand washing depends not only onthe regularity and thoroughness of the procedures used butalso on the type of hand-washing agent selected. The con-tinued occurrence of disease outbreaks, such as rotaviralgastroenteritis (9, 20), in health care settings suggests poorcompliance with hand-washing guidelines (1. 10). However,a high degree of compliance alone may not be suffcient forproper infection control if the hand-washing agent is noteffective in the removal or inactivation of important noso-comial pathogens. The findings of this study indicate thatsome commonly used hand-washing agents may be limited intheir abilities to deal effectively with viruses which areknown to cause disease outbreaks in institutions.

Most of the in vivo evaluation of hand-washing agents hasbeen conducted with bacteria; there are very few publishedreports in which the agents have been tested against viruseson experimentally contaminated hands (8, 13, 14, 28. 29).Furthermore, there is no standard in vivo protocol to test thevirucidal properties of hand-washing agents.

Whereas in commonly used suspension and carrer iestingof germicides a disinfectant is required to reduce the infec-tious titer of the test organism by at least 99.9% to beconsidered effective, there is no such generally recognizedcriterion of effcacy for the in vivo testing of antiseptics forviruses. Schurmann and Eggers (28) and Carter et al. (6)reported that antiseptics were more effective in vitro than invivo. In view of this, it is considered necessary to test otherformulations and additional representative viruses and bac-teria before deciding on a reasonable criterion for effcacy forantiseptics tested by in vivo protocols.

Although surveys on health care personnel have shown

that the mean duration for total hand washing is about 8 s(21), in this study, the 10-s exposure of the contaminatedarea to the agent being tested was based on the hand-washing guidelines of the U.S. Centers for Disease Control(12). This relatively short exposure time is also consideredmore suitable for testing antiseptics used for hygienic handdisinfection (21, 23).

The rinsing of hands may be an important step in handantisepsis; tap water is normally used in the field, and wefollowed this practice in the testing protocol. Although it isacknowledged that the composition of tap water may varygeographically and temporally, neither distiled nor standardhard water is believed to be more predictive than tap waterof the results which would be obtained under natural condi.tions. Moreover, the organic or inorganic content of tap

water could affect the elution -of the test organism from thefingers. It can be argued that the residual chlorine in tapwater plays a role in the inactivation of pathogens on

contaminated hands. We do not believe this to have been animportant factor in this study, as the residual chlorine in ourtap water was low (0.05 to 0.2 ppm (0.05 to 0.2 f.glmlj).Previous work in our laboratory has shown that rotavirus is

I:. ' .. ... .J . : . . ~ , ~

OL. 55, 1989 HAND-WASHING AGENTS AGAINST VIRUSES AND BACTERIA

:latively resistant to chlorine in tap water (22). Further-iore. the feces used for suspending the test organisms may.ive interfered with the action of such small amounts of:ilorine by neutralization of Ihe chlorine or protection of the~st organisms or both. However. inactivation by tap wateriuld occur for some other viruses (8, 22) and bacteria.herefore, we suggest Ihat dechlorinated tap water may be a.Iitable rinse water for routine in vivo testing of antiseptics,lit this needs further investigation.The selection of a human rota virus for this study was

,ised on several facts. Not only are these viruses among the¡ajor causes of acule gastroenteritis, but they are frequently

nplicated in disease oulbreaks, even when chemical disin-:ction and antisepsis are regularly used for infection control'0). They are also among the very few human pathogenicTuses whose minimal infective dose. as judged from humanJlunteer studies (33), has been shown to be 1 cell culture(.: ':ve unit. The viruses are capable of surviving on humanii. ~ for several hours (2) and on nonporous inanimateirfaces for several days (27). Infectious rota viruses can beansferred readily between hands and inanimale objects (2),id both viable viruses (15) and their antigens (25) have been:monstrated on the hands of care givers. Considerable/idence on ihe relative resistance of rotaviruses to chemicalisinfectants is already available (18, 30).E. coli was chosen as the representative bacterium be-

iuse it is a widely accepied indicaior of fecal contaminationid is also a common pathogen transmitted by hands.revious studies on antisepsis have used strains of this:-ganism as models (3. 24). The chal1enge levels of boih of

ie test organisms and the organiè load were realistic forJrmal hand contamination in the field. although it is recog-;zed that higher levels of challenge may exist from time tome.In a field situation. antiseptics should be suitable for the

:activation of both viral and bacterial pathogens on contam-¡ated skin. This may be particularly true when the minimalifective dose for some viral agents is extremely low.urthermore, some viruses may survive on human skin more;adily than many bacteria. In this study, E. coli was clearlyii susceptible to inactivation during drying than was the

l(e. ,irus since a smaller proportion of its initial inoculumas recovered after 20 min of drying on either finger pads orands. Recovery of both the virus and the bacterium fromie whole-hand surface after 20 min was less than when onlyie finger pads were contaminated. However, it is not known.hether this was due to the decreased effciency of recoveryr the increased rate of inactivation of the test organisms oroth.Use of the whole hand for regular testing of a'ntiseptics

gainst viruses is not considered feasible. Apart from the:Iatively large volumes of a high-titered virus pool requiredir each experiment, determination of the base titer of therganism and treatQ1ent of hands with the test product muste conducted at different times. For viruses, a detoxificationlisinfectant removal) step is also required. Moreover, re-overy of inoculated organisms from the whole hand iseneral1y lower and more variable than from finger pads.'hese factors make ihe protocol inherently diffcult to con-'01. On the other hand, the finger pad method describedere represents a simpler and better way of testing antisep-cs against viruses as well, as bacieria. It not only permitsie use of microliler quantities of the infectious agent but

Iso allows the inclusion of proper controls and suffcient. umbers of replicates in the same test. The results obtainediith this protocol were reproducible and did not show any

significant person-to-person variation; they were also inagreemenl with the findings based on the whole-hand tech-nique. The suitability of using fingers insiead of whole handsin antisepsis experiments has also been noted by others (28).Studies in progress in our laboratory have also successfully

applied Ihe finger pad protocol for the iesting of hand-

washing agents against rhinoviruses.Alcohols and formulations containing 70% alcohol were

the most effective in reducing roiavirus and E. coli contam-ination of hands. This is in agreemenl with findings of otherinvestigators (3, 24). In The Federal Republic of Germany.60% isopropanol is used as an index for comparing Ihe invivo bactericidal effcacy of antiseptics (24). Much of the loss

of infectivity of the virus and the bacterium on the skinsurface due to these aniiseptics may be the result of in situinactivation. In the case of the other agents tested, it mayhave been predominantly due to wash-off, as discussedbelow. Furthermore. the addition of emollients to alcoholsor alcohol solutions of chlorhexidine salts makes them lessastringent and more widely acceptable (17). Although bothHibisol and Savlon contain 70% alcohol. Hibisol contains ahigher concentration of chlorhexidine gluconate than Savlondoes at the recommended use dilution. Despite this, Sa vi onperformed better under the test conditions used. This mayhave been due 10 the presence in Savlon of cetrimide 'as anadditional active ingredient.

Deltol was chosen as a product containing PCMX. Theconcentration of peM X in the undiluted product tested washigher than is usually found in PCMX-based hand.washingagents (17). Like Deltol, Proviodine is an antiseptic notroulinely used for hygienic hand disinfection. However. itwas selected because previous studies of rotavirus disinfec-tion (30) showed it to be the most effective of the iodine-based formulations tested. Even though these products werefound 10 be more eO'ective than soap and aqueous chlorhex-idine salts, the level of rotavirus removal by the more dilutesoluiions of PCMX or iodine-based products formulated forhygienic hand washing may not be as high.

Aqueous solutions of chlorhexidine gluconate (Savlon andCida-Stat) are commonly used for hand washing; the resultsof this study clearly demonstrate that they may not bereliable antiseptics for pathogens such as rota viruses. In

fact, virus removal by tap water alone was comparable tothat. achieved by both dilutions of Savlon in water. Thissuggests that the reduction in titer levels by this product maybe due primarily to wash-off. Similar results for the action ofaqueous chlorhexidine salts have been obtained in in vitrostudies of rotavirus disinfection (18, 30).

The routine use of plain soap and water is considered bymany to be quite adequate for the decontamination of handsin infection control. This is not borne out by the resultsreported here. Washing of contaminated hands with almostany agent is clearly desirable, but it should be noted that useof a less effective product could actually lead to the spread oflocalized contamination over tile entire surfaces of bothhands (29). This phenomenon is under investigation in ourlaboratory.

Regular and proper washing of hands by health carepersonnel may be inadequate if ineffective products arerelied on. Therefore, selection of an antiseptic with demon-strated broad-spectrum effcacy is considered essential inconjunction with efforts to increase compliance with goodhand-washing practice. Proper testing of hand-washingagents against both viruses and. bacteria by an in vivo

protocol is clearly desirable, and the finger pad protocol

presented here may be a suitable model for such standard

3117

, 3118 ANSARI ET AL..' .~

tests. Further evaluation of the finger pad protocol withother viral and bacterial pathogens is under way' in our

laboratory.

ACKNOWLEDGMENTS

Financial support for S.A.A. has been provided by a scholarshipfrom the Ministry of Education of the Government of Pakistan.

Complimentary supplies of Cidomycin were received from Rous-seL. We are grateful to the volunteers who agreed to participate inthis study.

LITERATURE CITED1. Albert, R. K., and F. Condie. 1981. Handwashing patterns in

medical intensive care units. N. Engl. J. Med. 304:1465-1466.

2. Ansari, S. A., S. A. Satlar, V. S. Springthorpe, G. A. Wells, andW. Tostowaryk. 1988. Rotavirus survival on human hands andtransfer of infectious virus to animate and nonporous inanimatesurfaces. J. Clin. Mic:robiol. 26:1513-1518.

3. Ayliffe, G. A., J. R. Babb. J. G. Davies, and 11. A. Lily. 1988.

Hand disinfection: a comparison of various agents in laboratoryand ward studies. J. Hosp. Infecl. 11:226-243.

4. Black, R. E., A. C. Dykes, K. E. Anderson, J. G. Wells, S. P.

¡i , Sinclair, G. W. Gary, Jr., M. H. Hatch, and E. J. Gangarosa.v~ 1981. Handwashing to prevent diarrhea in day-c:are centers.

Am. J. Epidemiol. 113:445-451.

5. Blackwell. J. H., and J. II. S. Chen. 1970. Effects of various

germicidal chemicals on H.Ep.2 cell culture and herpes simplexvirus. J. Assoc. Off. AnaL. Chern. 53:1229-1236.

6. Carter, C. H., J. O. Hendley, L. A. Mika, and J. M. Gwaltney,Jr. 1980. Rhinovirus inactivation by aqueous iodine in vitro andon skin. Proc. Soc. Exp. BioI. Med. 165:380-383.

7. Casewell, M. W., M. M. Law, and N. Desai. 1988. A laboratorymodel for testing agents for hygienic hand disinfec:tion: hand-washing and chlorhexidine for the removal of Klebsiella. J.Hosp. Infec:1. 12:163-175.

8. Cliver, D.O., and K. D. Kostenbader, Jr. 1984. Disinfection of

virus on hands for prevention of food-borne disease. Inl. J.Food Microbiol. 1:75-87.

9. Di Matteo. A., A. Sarasini, M. S. Seotla, M. Parea, G. Licardi,

and G. Gerna. 1989. Nosocomial outbreak of infant rotavirusdiarrhea due to the appearance of a new serotype 4 strain. J.Med. Virol. 27:100-104.

10. Donowitz. L. G. 1987. Handwashing technique in a pediatricintensive care unit. Am. J. Dis. Child. 141:683-685.

1 i. Ford-Jones, E. L. 1987. The special problems of nosocomialinfection in the pediatric patient, p. 494-540. 1/1 R. r. Wenzel(ed.). Prevention and control of nosocomial infections. TheWilliams & Wilkins Co.. Baltimore.

12. Garner. J. S., and M. S. Favero. 19l15. Guideline for handwash-ing and hospital environmental control. 1985. Centers for Dis-ease Control. Atlanta.

13. Ha~'den, G. F., D. Deforest, J. O. Hendley, and.l. M. Gwaltney,Jr. 1984. Inactivation of rhinovirus on human fingers by viru-cidal activity of glutaric ac:id. Antimicrob, Agents Chemother.26:928-929.

14. Hendley, J. 0., L. A. Mika, and J. M. Gwaltney, Jr. 1978.

Evaluation of virucidal compounds for inactivation of rhinovi-rus on hands. Antimic:rob. Agents Chemother. 14:690-94.

15. Keswick, B. H.. H. L. Pickering, H. L. Dupont, and W. E.Woodward. 1983. Survival and detection of rotaviruses on

ApPL. ENVIRON. MICROBIOL.

environmental surfaces in day care centers. Appl. Environ.

Microbiol. 46:813-16.16. Larson, E. 1987. Skin cleansing. p. 250-256. 1/1 R. P. Wenzel

(ed.). Prevention and control of nosocomial infections. TheWilliams & Wilkins Co.. Baltimore.

17. Larson, E. 1988. APIC guidelines for infection control practice.Guideline for use of topic:al antimicrobial agents. Am. J. Infec:l.Control 16:255-266.

18. Lloyd-Evans, N., V. S. Springthorpe, and S. A. Satlar. 1986.

Chemical disinfection of human rota virus-contaminated inani-mate surfaces. J. Hyg. 97:163-173.

19. Ojajarvi, J. 1980. Effec:tiveness of hand washing and disinfectionmethods in removing transient bacteria after patient nursing. J.Hyg. 85:193-203.

20. Pacini, D. L., M. T. Brady, C. T. Budde, M. J. Connell, V. V.Hamparian, and H. J. Hughes. 1987. Nosocomial rotavirusdiarrhea: pattern of spread on wards in a children's hospitaL. J.Med. Virol. 23:359-366.

21. Quraishi. Z. A., M. McGuckin, and F. X. Blais. 1984. Durationof hand washing in intensive care units: a descriptive study. Am.J. Infecl. Control 12:83-87.

22. Raphael, R. A., S. A. Sattar, and V. S. Springthorpe. 1987. Lackof human rota virus inactivation by residual chlorine in munici-

pal drinking water systems. Rev. Inl. Sc:i. Eau 3:67-69.23. Reybrouck, G. 1986. Handwashing and hand disinfec:tion. J.

Hosp. Infect. 8:5-23.24. Rotter, M. 1988. Are models useful for testing hand antisepsis?

J. Hosp. Infecl. I1(Suppl. A):236-243.

25. Samadi, A. R., M. i. Huq. and Q. S. Ahmed. 1983. Detection of (j"

rotavirus in the hand washings of attendants of children wiihdiarrhea. Sr. Med. J, 286:188.

26. Sattar, S. A., M. K. 1jaz, C. M. Johnson-Lussenburg. and V. S.Springthorpe. .1984. Effect of relative humidity on the airbornesurvival of rotavirus SA-II. Appl. Environ. Microbiol. 47:

879-81.27. Sattar, S. A., N. Lloyd-Evans, V. S. Springthorpe. and R. C.

Nair. 1986. Institutional outbreaks of rotavirus diarrhea: poten.tial role of fomites and environmental surfaces as vehicles forvirus transmission. J. Hyg. 96:277-289.

28. Schurmann, W., and H. J. Eggers. 1983. Antiviral activity of analcoholic hand disinfectant. Comparison of the in vitro suspen-sion test with in vivo experiments on hands. and on individualfingertips. Antiviral Res. 3:25-1.

29. Schurmann, W., and H. J. Eggers. 1985. An experimental studyon the epidemiology of enteroviruses: water and soap washing

of poliovirus l.contaminated hands. its effectiveness and kinei.ic:s. Med. Microbiol. Immunol. 174:221-236.

30. Springthorpe, V. S., J. L. Grenier. N. Lloyd-Evans, and S. A.

Sattar. 1986. Chemical disinfection of human rota viruses: em.cac:y of commercially available products in suspension tests. J.Hyg. 97:139-161.

31. Steel, R. G. D., and J. H. Torrie. 1960. Principles and proce-

dures of statistics. McGraw-Hili Book Co., Toronto.32. Steer, A. C., and G. F. Mallson. 1975. Handwashing practices

for the prevention of nosocomial infections. Ann. Intern. Med.83:683-90.

33. Ward, R. L.. D. i. Bernstein, E. C. Young, J. R. Sherwood, D. R.Knowlton, and G. M. Schiff. 1986. Human rotavirus studies involunteers: determination of infectious dose and serologicalresponse to infection. J. Infecl. Dis. 154:871-880.

ør~~::,'''~~::t''~;.:;:.j~t7~T\~~~/''~'.'f " ;.;),'. .~~~:.?~1"'. ::::;;'.~.:.: . ',' "', .' "'.:. .., .' , .", . ~

iSurgical Scrub and Skin Disinfection'::,aha-n A.J. Ayiiffe, MD, FRCPath

(

(

(

ABSTRACTThe role of pre-operative disinfection of the sur-

geon's hands and the skin of the operati ve site in theprevention of wound infection remains uncertin. Thenonnal resident skin flora. consisting mainl)' of coag-ulase-negative staphylococci and aerobic and anaerobicdiphtheroids. is an uncommon cause of infectionexcept in prosthetic operations. Staphylococcus aureus is

rarel~' a resident on normal skin other than the peri-neum. and is mostl~. present on the hands as a transientacquired from tlle nose. Neverteless. it seems rationalto kill or remm;e all transients on the hands of thesurgeon and reduce residents to low levels.

Surgical skin disinfection is usually assessed b~' mea-

suring the reduction in organisms on the hands imme-diately after disinfection. after repeated applications ofthe disinfectant and after wearing gloves for two to

three hours. The hands are commonl)' sampled in abowl or plastic bag containing Ringer's or a similarsolution and relevant neutralizers. or by the use of glove

washings. A standardized technique is necessar~' toprovide a statistical comparison between agents. Anti-septic detergents. chlorhexidine or po\'idone iodinesho"" immediate reductions in bacterial counts of 70e¡rto 80c;. increasing to 999c after repeated application.

Hexachloraphene and triclosan detergents show alower immediate reduction but a goo residual effect.Sevent)' percent ethyl or 60Ck propyl alcohol. with or

without an antiseptic. show an immediate reuction ofover 959c . and in excess of 999c on repeated application.Residual levels of organisms tend to be lower afterrepeted alcohol tretment than following the use ofantiseptic detergents. A two to thre-minute applicationof the antiseptic to the hands, without a scrubbing

brush. is commonly reommended in the UK. but possi-bl~' in routine surgery' an application time of 30 seondsis suffcient. killng or removing trsients and superf-cial residents.

f\.

(..

'.

fi.,,. UP 1),1'(;1"'1" . "I .\I"Î1((lI.\lln"hl"iiJ:.~.lilt"'lIli,tIl! l',,,;.I',,II\. fliiffHt1\.tJï;, 1,1!,'I'lllii, h', "'II"¡1 l.aho"""'ì. !JUlIlI'i Roil''' HlIlllffll. /:,,mitlt/w11.¡ iiif,,: hiii!!"'."';

:;,;.:.". "p,,,,- III;IJI"'- f.. ("U),01li ."j :~\lllll. ,\l/J. Hr".lUlw' ¡"¡"rlII'"1\'.\11:-,;' 1.lihIIJul"'¡, liiull,~. ¡"Hltl HII\/JlI1ti,llntllli/t;,ti" HI,\ ';t.JI. i'lIlrd¡. ni~~.:""/

/'.FFLJ/()\ (.i\TR()l. /'l.,-l I."i. ' S, /

Pre-operative bathing or showering of the patient hasben shown to reduce post-operative sepsis in severalstudies. Althõugh repeated total body bathing withchlorhexidine reduces the number of organisms on theskin. it is less reliable and effective than the usualimmediate pre-operative antiseptic treatment of thehands or operative site. A recent study in which patientsundergoing general surgery bathed pre-operativel~' onone occasion with an antiseptic detergent showed noreduction in wound infection when compared with sim-ilar bathing with non-medicated soap. (Infect Control1984; 5(1):23-27.)

INTRODUCTIONFor the purposes of òisinfection. the skin Hora ma\ bt

òi\'iòed into "resiòents" and "transielis."1 'n1t residtl1flora colunize the skin and are remoH.d with difhnill\ b\usual washing procedures and are renio\td incoiipleltl;b\ most disinfectants. Resident fiora consist mainh 01

coagulase-negative staph\'lococci. micrococci. diphther-oids and Propioniho(/lT (In/ts. Other organisms. tg.Siiiphy/I/ll((/I (//ll'lll.l. ma\ colonize certain area, of Inl;I;.1skin as well as damaged or abnormal skin. On tht otherhand. the transient fiora. ego intestinal gram-negatiH'

bacilli. do not usualh' colonize normal skin and are l1oreeasilvremoved than .the residents. Since the compositionuf the skin Rora in an indi\'idual is rareh known. pre-

. operative skin disinfection is expected to remO\.e or kill-transients" and as man\' of the resident Rora as possible.

and should show an effect for at leasi three hours. 'f1t'sUl!~ical hand scrub has been defined tor legal purposesin the lS as "a non-irritating antiseptic aiiimicrobial-

containing preparation that signifìcanth reduces thenumber of organisms on intact skin. A surgical hand-scrub should be broad-spectrum. fast-acting and per-sistent. -:! However. such definitions are of limited use asthe reduction in organisms on the skin required lU reduct'wound infection is unknown.

In this anicle. methods of sampling: agents used fordisinfection and the meaning of tests will be discussed.The role of the skin Rora in wound infection will also beconsidered.

SAMPLING METHODSAlthough not necessaril\' tvpical of the skin of other

parts of the bod\.. the hand is a con\eniel1 model for

~:~

TABLE 5EFFECT OF NEUTRLIZER IN SAMPUNGFLUIDS FOR POST-DISINFECTIONSAMPLING ON THE TEST RESULTS WITHCHLORHEXIDINE GLUCONATE (CHX)

Neutralizer- inSampling Fluid

Log Reductionx :: .abseni 4 S 05=.

presen~ 30 05::

. :-"V :e! 150 m' Tween 80 - '5 ç 'ecitnme - :: 9 nisridine

.. 2 c ' 007

Dismfecrion 1 mm hanO-wasr wirl" CHX (40'0' derergemTesr bacrenum Serrai,,, marcescens .ATCC 30127Samplmg metnod giove IUlce merhodDesign of srud\' crOSS-Qve.' 16 volu"lreers

lectio,i wa~ heiweeii 4,1 and -t.::. Tahlt 6 sh(l,'~ ihai pro-cedures based UPOIl nibhin~ appropriaie disinfeClalisonin ihe hands are heiier than washing prucedures,

Excepiing ethanol ai ;t conceliratÎon of tlClÇí Vl\ allsuhstances sumniari7ed in the first group are at least notsignificani" interior to the et'hcan ofisopropanoI6OC;; \\. our standard. Among the three akohol~ there exists adear range of order a~ tar as eflican is concerned,name" n-propanol ? isopropanol? ethanoL. With E, ((l¡a- ;t tesi haneri u m a waten solution of pll.idone-iodint'with I (,~ availahle iodine is about as eflerti\'e as iso-propanol ()w,~ when rubbed onto the skin.

.-\11 washing procedures ,,'ith disinfectani deiergent aresignificant" inferiur to ihe siandard. In lac\. the". are notor hardh beiier than ihe degerming acti,'ity of ordinal'.liquid soap. In ihis toiiexl ii is imponant to realize thaithe efhcan of povidone-iodine together with a detergentis much worse than its counterpan, the wate~ solution.

In summan., our findings demonstrate that disinfec-tant detergents a\'ailable ai present do noi fulfill iherequirements of h\'gienic hand disinfection. not onhbecause the" imply undesired washing procedures. hut

also because their degerming acti\'ity exceeds hardlv thaiof ordinal" detergents. So. we regard alcohol in appropri-

aie concentrations as ihe most suitable substance forh\'gienic hand disinfection. ai the moment.

REFERENCES

IXuische (~esells(.hah fur Hv¡:ient' lind Mikmhioloi;ie RichiliiiienliJr die Prütuii¡: unel I:e"erlun¡: dieniischer Desiilekiiom,.ei-lahreii-Ersier Teilabschniii. Zb; Bilki Hi/(. I. ...hi Uri¡: B l\llll:i';~:;J:!l'.~5ti,

.) OslelTichischt. (~t'sellschalt lUI' H,fo1ene. ~fiLrohllllllfole unel lrä-vt'lIimledizin: Richiliiiie ,.oni .¡. ~t)\.eniber l\llllIl ur dlt' B('t'nun¡:del' Oesinft-LlioIlS"irkulili \On \'erlahren t ur die H, Ilit'iischt.Häiieleelt'sinkkiion. (j,ln,nr/Ii.,/¡. l\iriilt,.iill1.I':"'IIIIIr. I\I~I:~~:~:-\.:H.

.).)

TABLE 6EXMPLES OF THE EFFICACY OFVARIOUS DEGERMING PROCEDURES ASASSESSED BY THE VIENNA MODEL

Procedure. Conc Log(all 1 minute) % Reduction

Rubbing-in

n-propanol 60 Vlv 5550 V'V .: 940 V,\¡ 4 .,

Isopropan.9! 60 v'v .: 2

ethanol 80 v'V 4 ~

70 vv " 0

60 V'V 38'

povidone-iodine solulion 1 g.ii 40

Washing with

povidone-iodine soap 0.75 gv 32'

chlortexidine soap 40 g'v 2 g'

phenolic soap mixed 29'

liQuid soaP ÒAS 9 20 vv 3 C'

. Sigmficantiv less effecrive than srandard

Values denved frofT different exoenmenrs bur fro,. one :aoorclOry.. the mean log reduClIor of stanaard dlsinfecuJ" a'wa,.s oe''';;4 7 and 4.3

:t Led.. \ E: lJlr \r".",., .\I"di:"u.,rh,. ,'rlml, 1m /4 ¡f/iliil/",tI,.! (,1..:.

Biihlaus "a( hI. l'-i:;. I' ~i~.4. l'dchner F: Zil tn',,', '\iiI,I: . Zn',,'ii Ii"iim If .\,."".1"",,, I'I,IC:"

Suiini zdra mlii(.Le 'akladalesl\1. I !-eW. I' 'ill

:;. Rt"elanioii: Ho('hsi wKhii¡:t- Erlahruii¡:eii Ubei d't. .\lic,IIii:IC nt., iii(~bäral1lali('nepldt'nilscheli PUl.rperalhelX'r Ú'II,dirIi ner ~...C,..IL"ha/1 d" ..h:.lt.:11 \\'''11 1~411: '¡:2'¡~-24'¡.

6. Semmt"';.t'i~ IP: Die Aihiologie. del' Begrilt und elit' I'n.pll\l;i\,I-dl.~ "'ind~iifi('~rs. Wit"JI. Harleben~ \-~r1ai:. I~iil

,. Coh('n J: .\1011.1/(0/ p"u." ...iiah..".i I'" tl" B'IIUI',,,ml _\"'.11".'. 1'(';..('d. !\(' \ürL. Academic Prs~. I!-ïï. pp lï\l-21:t

1' I'undi M. 1'011('1 W. Miiiermaver H. ("I al: Thoui:hi, UpOIl iilt'construciimiof llquir(Olt"ntS of disinleClion proedun', iiiiin~rniil\:contaminated hands. ZM Bolit H.'l!' I. ."bi ()n~ R l\lï3: ltil: Iti.1-I-:-:

9. Rouer M. MiliermaH~r H. Kundi M: Im.esiifoaiiiins lin iht, oiiind 01ih(' anificialh contaniinait" hand-Proposal of a It'SI mt'ihiiel Z;';Boltl H.Yr..I. Ahi On, B 1974: 159:5611-51'1.

10. ROlI('r 1\: Handed('sinfekiion. in Horn H. lrimr I. Weiifteii \\(('dsi: Hoiidhiirli dri /),.III,ItI/(1i uud S",./i\(iiiii. \'1. ,.. Kerhii. \'lBVerlag \olL und (;e~undheii, 19R:~. ml 5. in pllS'

i I. lS General Services .Adininislraiion. O. T-e dru¡¡, ¡i.-nt'ralh rt'l("~'nized as safl.. effeClin' and not mishrandeel- TeOlaiiH' hiial mde!.('S F,dnal Rr¡rlltl I':;~: '¡3(41:1211-12~9.

12. Roiier ~l. We"õlILaC. Koller W: InAul.nt.e 01 ~iini.. ,õlriahlt., 1111 int'llSUII' of t"õlluaiiom 01 procedures lor h,¡¡iem, hanel disinlt'cii"il.HYp:rlif .\1 ttli:ii i \11' I: ï: 1 5 ï - 166.

13. .-\1\ R. !\laihach HI: COOlparali\'(' siudi on the aOliiiirriihial ef ie-iiof 0.5(,; chlorhexidine folUnmaie and iO" isopriip\l alwhol 011 iht'normal llora of hanci~. ...ppl bi;'/I", .\lm"h",¡ I\lï~: :~i :tilO-tii;;

14. Ah R. ~taihach H i. .-\ comparison of tht' aniimicrohi..i etlt'C "i0.5'; chlorhexidiiw ('Hibi~iai'i and ï(l'; 1"'-"1'1,01111'1 oil h.mo.

coiiiaOlilialeel i,.iih S. maln',(f" '. L/ii f.,1' Th 11""'..1 I\lXli, 'i: I\li '~"i.

Ho"ri tJl"rrlrrlw,,!Ri':ll'

11' 12.6

20

99,2

Chlorhexldlnedetergent

(4%)

26.4

30

40c:o-~ 50~.... 60

70

8087.0

90

100Bar SOD

~ 10DDlicotlon

~ 6ooo1lcotlOns

68.0

98,0 99.799.7

Po Idon-Iad Inedetergent

(10%)

Ale, chlorhe,(0.51)

Figure 1. Surg1ca. nanc o'sir,ieCllon

comparing ilit" effeci 01 r1itlnenl disinle(\aii~. Hand~ artt'.i~ih ,.impled. Iianerialiouii~ art usualh hi~h. laliora-111"' q.d! iaiilit. collenienth useo as iest sul~ie("s. andsiaii,ii, aliesis m,i\ he applied to rebii\'eh small nuiihenOllt-'I' How('\.er. pailiogen~ on normal hanos are le\\ anothe iiiiriiai flora i~ u,ed lor comparaiive iesis. For ihi~n:a..' ilL. Lan- must Iie taken in interpretiiig the resuli!o inlerm, cit possihle pre\.eniion of inlection.

Tes!' usualh inmlve sampling of ihe Iiands of a num-her of subjects and couniin~ ihe organisms releasedht'i .re ,mel aher disinfection. The hands ma\ be rinsed ina bowl'''l or a plastic hag"' containing fluid with an appro-priaie neutralizer. or the hn~enips ma\ be sampled b\ruhhing them on the hase of a petri dish containing asimilar tluid.~"" Counting organisms in glove juice orwashings is a Ilethod lür mea..;uring the persistent effeciof the agent.7.I" Contact plates are less efficient. but areuseful lor in-use studiei;.~' Other methods are sometimesu~t'd and ha\'e been re'iewed elsewhere.

ii. I i Although

there are dinerences in results obtained with these meth-od~_ the comparative bacterial counts from the differentsampling methods are reasonably consistent.

i:!

COMPARISON OF DISINFECTANTSDisinfection of Hands

\Iosl of the agents used produce a significant reductionin bacterial flora when compared with washing with soapand \,'ater. Fi~urt i sh(),'s results obtained from studies inBirniin~ham.i:\-i-, When using a detergent preparation.the hands and wrists were initiall\ moistened and 3 ml to;i il I of the product were thoroughly rubbed in for twOminutes. The hands were then rinsed and dried. With the

:!~

alcoholii preparation. 5 ml were poured iniii nippedhands and \'igoroush rubbed m.er h;uids and \'Tisi' 1I1l1I!dn. -nie pro~ess \\'a~ then repeated. .-\koholic chliirlie,.idine was the most eflecti\'e following a single appliGitll 'I..but pm.idone-iodine and chlorhexidine. altliiiugh iiii-tialh less eflecti\.e. show a similar lUllulati\e elleei lIllrepeated application. Hexach lorophelH' also ,him ~ .111

excellent cumulative enect. but is no\\' le~s commoiihused. Irgasan is also similar in acti\.ii\ to he,.achlorophene and has replaced it in mam prt'paratiombut. as vet. is not commonly used as a surgical scrub.

i ¡

A continuing effect over several hours ma\ be an impor-tant propem of a surgical scrub and can be demunstraterby sampling the hands after wearing gloves tor threhours. Figure 2 shows that chlorhexidine detergent andethanol or isopropanol. with or without an added disin-fectant. show a persistent effect. It is surprising that alco-hol shows this effect. but is probabh. due to the wntinuingdeath of alcohol-damaged organisms over the three-hourperiod.lri The persistent effect of povidone-iodine israther less than the agents shownY.I' Although it is re.ognized that some operations mav last longer th¡in thrthours and organisms initially suppressed ma\ shil\growth ai six hours.lt' three hours is a rtasonahle time forcomparative tests.

Disinfection of the Oprative SiteThe results obtained with the hand disinleciion experi-

ments may be generally applied to operati\'e siie disinfec-tion but the maximum effect is required un a singleapplication. For this reason. alcoholic soluti()n~ art ah,"¡\ spreferrd.. Table i shows the effect III ihree agents on the

.\U.¡:rl/.\kii /11.'11".((1''11 .~ii¡(,

10 J12.0

i

n201

17.0

~30 "i

\i¡0 ~

c I I050 J-

':î

i.... 60 ~

70 Ji!

80 j

90

100Bar SOOD

87,0

ChiornexioineOetergent

Ale. chlornex,(701 I sooronvl )

701 I soorODV i

Figure 2. Su'g.:a .,arc ::'sinfectlon

,i.in lit the ahdiiiieii using contact plaie.. lor sampling. I"

Pri 1\ ¡dec! an appn i\ed alcoholic solution is used. there i~illllt, t(J ihii(Jse bet\,'een ageni~, The mode of applicationII! thl: disinfectant mav be import.int: nibhing in an ako-Ii. ,Ii(a!.eiii \'lth a glo\i"d hand was sigiiificanth mort'et ienì\.e than using .gauze.:!Ii '. ,Pre-perave Bathing

Bathing or showering with ~oap and \\'ater is associated\,'ith an apparent increase in organisms on skin. whereasa reduction is obtained with antiseptic detergents.especiall\ chlorhexidine.:!l.:!:! Chlorhexidine detergent isa~ effective as hexachlorophene in reducing the skin floraand would be expected to have a similar effect on woundinfection. Cruse and Foord:!:\ showed that pre-operative

bathing \\'ith hexachlorophene was associated with a lowr\\Qund inlection rate than bathing with non-medicatedsoap. A lower staphvlococcal wound and nasal coloniza-tion rate was also shown by Brunn:!~ following the intro-duction of hexachlorophene washing

and nasal pro-

ph\.laxis with fram\'cetin. A reduction in infection rates ofgroin wounds from i i .5'i to 8tì was obtained in patientsundergoing \ascular surgery: the patients showered threetú eight times. \,'jth chlorhexidine detergent before theoperation.:!:' Howe\'er. a recent stud\' in twO hospitals inBirmingham showed no difference in infection rates ofclean wounds (Table 2) follo\\'ing pre-operative bathing\'. ith either chlorhexidine detergent or non-medicatedsoap,:!" It \,as onh possible for patients in this stud\' toha\e one pre-operative bath and it ma\, be thai chlorhex-idine would have shown a greater effect aher repeatedbathing or showering. ~evertheless. bathing with chlor-

\;.:;.l ~'I('\' (J\-lH(J!. Jl.."""' \-iii .; \" 1

~ 1 treatment

~ ofter wearIng gloves for 3 hours

TABLE 1

DISINFECnON OF SKIN OF ABDOMEN

Percent Meen Reduction inBacteriel Counts (25 SC em)

't hoursImmediilely efter

Agent After Disinfection Disinfection

70% Alcohol 99.7 iii 89

o S% AicoholicChiorhexidine 99.0(3) 77 6

AlcoholicPovidone-iodine 99.9Ii) 913

i ) = count per plate.

hexidine detergent is an effective method of remö\'ingSiaph. aiirl'lLS from the skin:!'; and would be valuable incertain patients, Table 3 sho"'s the remo\-al of SllipJi niirrwfrom the skin of a patieni with a colonized bed sore beforea hip operation.:!'"

CHOICE OF SURGICAL SCRUBPrferablv. the choice should be based on clinical trials

of wound infection rates following the use of the \ariolla~ents. but the limited a\ailable studies do nO! show am

9._...

TABLE 2

PREOPERATIVE BATHING WI SOAP OR CHLORHEXIDINE DETERGENT IN TWO HOSPITALS

AgentU..d fora.hing Tot.1

Cle.nNumber of

33:'! :

. 9E

:: t .;: ~= :.:J:.: ü

, 872:: 3,

. 74E

,:; 9'

362C136'

,.C".. _. "::: _.:;~~

Cr'- :0_-= i :"'f:::?4?: 6

:i",'.;':i~"

";C'. ~':5~,~

:: 3

676199

3E','; :i

Wound Infection RatesCleanl

ContaminatedOper8tionl

CleanlContaminated

(Percent infected)

,'l

:;3':,~o 7.

1011\ iil. lit. rlilkrt'llll". :\11 il1prllt'cl Ili'ihiid III ..kiii dis.

iiiltiiiiii\' ii,iii~ 0.:1',; all iiholii Ihliirht'xiclilH' 1111' ihi'

II\X-r;iiiiiii siit' alll ht-adilo1"lpht'nt' 1111' h.iid.\\eI..hing.\\.1' ;1'..'.. .ialld wi ih a rt'cl u(" 1011 iii ¡iii..i.iiperai i\t, st'psisIriin: 47'. iii :~.II';. II iht' Binniiighaii :\lliclt"1I1 Hiispi.

1;11. hii: 'lillll I h¡lIgt"' iii prt"ciiii\t" lleaSUI"" c..nlrn:cl;11dll ..,ii:ic tllIl', II '0 sii:nilKall clillt'I"'Ilt" iii inlt'"liiiiiral(" \\t'II' Iihl;lilllcl iii ihrtt' oiht'r "llldit', iii whidi iiiii.pari"'I1' 'l('I\\t'I'11 a~t'III' \\t'rc- in¡ifk.~.;'::" hui l\\11 oflht'Sl'wi'a ""1 illiiriilltcl iriak Iii ihi' Irial111 Bt'I1'\ ei al.'III

i hi'i"t \, l"n'si~iiil K.iiii h It'''' I1ltet iiiii, ii dt'aii 01ltr,,1 ioii,aiid it'' "i"pl" It IUlcial inlt'l"ilili, \\'lt'll ihltirht'xicliii'W.I' ii..ed lor hoi h su rgical ;.uh aiicloperall\t' ..iit' di..lI-itClIllli ihan \\'iih ..imilar u..t' wiih poùdont'.iodint'.How~er. ihi' numher! were raiher ..mall. inlel"ioii raieswt're rai ht'r hil(h ancl thelT were disc:n.'pancie.. 1t.'J(, tht'inridt'Illt' of in!tCI ioii in hernia operai ions \\a.. nollTdUled.,

The ihoil.t" i herelcire must he hósecl on lahoraiol" SI ucl.ie~. hui ..ome prcihlem.. ~main, Riillt'r ei al" l'omparedprcidun~ wiih 6OC;; ,.prcpaniil applied fill' hve minuie..ann ~hlJ\\'t'cl ih;u all deteriient p~paraiions were si~ifi.caiiih It...s elïi'dive. ROlier also l'nsider!o ihai ihe hrMappliiaiion should be effecii\'e and aqi;ue!o thai tht'nimulaiive efl~I'1 of repeated applicaiiom is irrl~'3ni.

H(J\\~er. ihere is no clinical C"'idence 10 suppon this

iipimiin. and mail' suqi;eons preteI' 10 wash iheir hand..

with running \\eiier."l1e mt'ihod 01 applicaiion of the disinft'cianl. ihe timt'

~ptnt on hand disinf(-tion and ihe meaning of ihe iesi~are al;;o wonh consideraiion. Wound infections caused h\iht' rt..idt'11 skiii Aòra are f('. In the 'aiional SUT\'t' iifInlt',iion ian-it'd OUI in ihe li' in iy~iiii onh 5~;¡ 01

",(iund IIleClion~ \\ere associalt'd \\.iih foaiiula..e-neiiaii\,t'slaph\I'K'1( ci or micrococci. InteClion,' cau..t'd b\ ihe..eonrani..m~ art common folluwing inst'riioii of a pro...i1W'I', hui in iht' rect.lllh lTpoiied lariie irialof hip and"nt'(. ¡o1l1 pnlSllit'ii( suriit'I'\, mo!tol ihl' iiiltiiiiin~ wt'rt

:!t

01 airhonit' oriiiiii. .I:! Howt''er. ."il/iilt. filII I'll \ i, Sl ill ;i i 11m.

moii iaust' 01 \\ound inlenion, bui ¡, r.lI"th .1 1"t..ickJl 1111normal skin apan InJll ihe periii'um. I ii ;1 sii(h .11di..inlenioii 01 nUI',e" hand.. u!Oili~ ciinl.KI plail", It; ill~t,:! (I,r; i showt.d ."Ulph. 1/1111.11\ in small Illlmht'l', heliil'l'

in,'ólimeni and onh i 01 :!i~ i(),:~.".;, ahl" ap¡ilil.tliOln 01ill'" eihd alcohol 1m ~1I..t'riiiik:l\ Siiiil.irh, iiiiht, ,¡(ufh

ill disinlt'rtion of iill ..kin 01 iht' ahclonlllI, "fi/'I' 1;'"'.11'

wa, Immd in onh 401 :!:~l' (I.il;;) paiii'ni, ;iiel 110m'

"i ~i:!

Mall Ilemher!' atier cii!Oinft'clion. il1~..pt'('1 iw ,it \\ ht'11i1'1'ihe disilllt'rtani wa.. fon..idl'rtd eflt'l'ii\t' III' nol.I" ~,iiih.hin~ lor hvt' 10 It'n mimllt'... \\.iih 'oap aiin li;tll'l' I' II,..,elïel"ii\.t' ihan aii antisepiir \\a,h UM"cl 1111 11111 niinuii"

wilhoUI a hrush.l:! 't''eiihelt"s. ii is impiiii.ani ihai "iisurlal'e.. 01 the hands ai~ adei.uaieh io\crt'cl' i aiicl adehned hand \\eishing iechnii.ue should he iau!ihi. \\llt'llassessing ihe effeCii\'eness of an a!(ent, ihi' numhi'r' 01

organisms remaining aher ireaiment mu,,1 al~ i he ciin,id.e~d" ..\ log 2 n:duction on the hand.. aht'1' di!Oinltcliiinma\.lea\,(' log 4 oriianisl1s, SOI1t' 01 \\'hidi (;11 ht'rt'IIUl\I'db\ funher sampliii¡i,

COMMENTSTh~ \'an'ing results are dilfïtuh \C iiiierprt'l aiid

depend on mail' faciors. ego ihe de¡iree 01 siandardiza-iion of the test. the ainount of \\'3ler u..ed in iht' deier!(ent\\'3sh. the type and conceniralÏon of alcohol 1!-lI.: eihaiiolseems \0 be as eflecii\(' as illt; in ..omt' siud¡t'~ bui niiiochersi. ihe mode of applicaiion iakuhol rubhed in i..more efleni\'e than rinsing or spra\'ng aiid alli)\\'iiig IIdnl. Akoholit preparaiiuns appear \I he mort eflt-Cliveihan the detergents after one applit¡iiioii and dilorht--idine ha.. a beltt'r residual efleci ihaii piiddllllt-iiidillt':bui possibh lilr lTasons alread\ di..ni"t'cl. iht' diiiiit' 01nne aiieii raiht'r ihan anoihel' is oheii dt'ridl'd b\ ii,acceptahilit\ raiher than on ihe n",u\i, 01 1;lhol'aIOI,\ 11.'1"'.The preferrd rhoii.e ¡, prohabh a ininii 1l1lldt'ralioliwht'll IOl1part'd \\ iili oiht"r larlll1". t'~" slirgKal iedi-

\'/';:" ", '''-11'1)''1'1/1' ,','"" ..i\;,f"

TABLE 3

PREOPERATIVE DISINFECTION WITHCHLORHEXIDINE DETERGENT OF SKINOF PATIENT WITH PRESSURE SORE

Number of Colonies of St.ph.per 25 sq em

Site of SamplingRight leg Front

below knee right thigh

.ureus

Backright thigh

;"':')vve'o ':¡, 36

-:-'),/..-:" ., ê :J::

. .~_. "'::.ut:;

'_.'oF" c c' o

iiiqiii', l\Pl' aiid ~iie olopl:r.llioli. ai.t: 01 palit:III.:I-' likely to

i:illueiice ihl' Cle\eli'piiit'iii ot.i \\.ouiid inleniiin,,\iilimigli PIT-opt'raliH- haiid disinltciiiin is a rationalpniii'dure, it ma\ he ihai .in \it"\\ III ihe small numbers ofiiathi i~eii~ I III IH 1 1"ii a 1 ~liii aiid iht" In\\ incicieiin.' of inlt-c-

~ioi! I au~ed h\ the iil11"iial 1I1lLI in general surge". a11\ !.li'lIl1 li.ind disinlli.iioii Ileihiid iit'.thoroiigh applira-¡1I111 01 ,igent Illr :~(l ,t"(lllid,1 \,oiiid hi' as elleCliH" iii

:Jre\l'lItiiig iiifectioii ;i, the usiial. more proloiiged pro-~ediin'.'"

REFERENCESl't It c PH' Illl" ¡i.ll It:rii,ill~\ 1)1 111.1'111.11 ,lII1: :\ IIt"\\ yu,llIllta11\( ll...1lipplll'd III ,I 'tiich 1.1 ihl" n,lIli'li.d 11111",1 ,lllillht"ch4ilnli'i tani .Kthlll III11li'ch.IIIU.tl tk.I1II1I~..III,f".' /)1. i~n~: li:L:~lij.:ql'.~."'III.iiil1 HI. ..\ iii ""11 re 1111.11 In:III..."l' "'1,1''1-, iii \I.iih." h HI. .\hlo It'd..i: .\A'iii .\111 ,,,hiii/,.~i. lú/i-; 111111 II' (.11111''; 1"',,/11111. 'l"\\ YClri.,

"'l'lililit'l- \ .l'r1.lg, I'll" I. PI' I :1,',- 141'.:\ I.."diiil' ~.. i. S~iii pn:par.llIon 101 "1"',...,''" 1"111/11'/ ill HIi,/Ii/1i1

.\I,.flu ",, pl'7:t III:l):.; ~',:~-I.4. (..""1111 \1. ~.I(Ik niiiip.iraii\t' ik ikii~ 1i,'lh'HIl- ilt-,lim'," aii

fHi'\alllI"lIl ,"'I.. 11.11.111111 rc .h,t'iim' d,', 1I.1I11-. H"¡, LIll :\rlii I'ii'":\:' ::~ 7:..:t 77.

.J l)¡iii,d". (""1,11" hall hii H'!CIl'lIl' IIl1d \Ii~rnhi"i"gi,', H/rliiliiiiriiFIi"J" /JtUflii,.i: ""d H,..i ,.,.¡",,!. ,-/',.",1,(;'1,,, /),\1I1It.,ltllfJII,oi'I'J'lalt".".tr.ii'i .\t'ilali-, h 1111 i. Si uliloal1 , (;ii,ia\ Fi..hl'r \,'d.ig, l'I~1.

ii K,"i('r \1. h.olllr W, WI'\'õllk.. (;: I~I\ Idom'-icHhlil' ami dilorhl'xidiiil'

J:h1li Hl.llt'.( (JI11..il1il1~ dt~lt-r~t"III~ lor ciisink.."lIUI1' CJ( h;.lid~../f#"'-""1ill HIi,IIiWIIII/I."'1I1I 1!11"1t: :!: H~-I;11"1'\'11'''''11 .-\F. Ko...iihlri. ,-\, .-\Ialal' sn: Coiip..raii'l t'.aluaiioii 01,"r!tii .1\ "riih I'ltparaiioii,. .'"1)! (.""ml ()/""'i l!l~": l-lii:ô:\.ô;,.

, Ihiidi \1: 'i'wi'r Ili'rnii,idl": \\liaiiht' ollti, iii \Iailui:h HI..-h K

It"d,i' .,'k", .\1" If,iulilo.I!;, RrI,...""u I,, eiiiuiil /"".("/111. :\c:\\ ,"orl...priigi'i-\crl;ig. 1'11"1. PI' iu:i-ii:!

" "illll1iI' lit.. 1.0 g 1\' JRC. Smiili (0: i:lll1l'hi,liht" III Hih"lltlh. H.

.\Inl l I'I~:\: 4 :~il'l;-\~!I.I II. ..d\,.\ II s, Ell" H. S~iii hanl'ii;i .ii,d '~1i1 C\i'III',.lioli lt1 "",idi'r..ci.

H' .\Irrll11I7:!: 1:1:11;-1411.

I: I.Clwh1i1' Ell. SIK',ial p,.Clhll'l1' II hli,pil..1 "IlN~V,i" iii Ku.-l'lI-\1), H\I~(1 \\"I:~. .-\\Iifti' (;.-1 IC-Ò'I: /)1,,,1,.(/'''11. IJ,i,"I-"'(lI"''' fili!"",iii,ii/i,"" (hliird, KI.1i kw~li. I!l":!, PV :!ô~,:!1"4.

/\Fr.lT!().\UJ.'THU/ .''''-l \" \

I:. H~11I R'I)l~l.(T111111l: ihe h.uid..i)i ..uig(.liri...iiid 11111..(" l'IC"'CI1It"rl,11thc: Iniernt.lilU1JI (,tll)~Tl""''' ilIlet ."\l1lpcl..iillll 1111 I'llililc.1I1' ¡II 1111.(.uiiirniol li(J"plial Iiiln.llllli. L.niulilll. iqxii. N"",-. \",,1'\ iii \I,d,nw' 'l.nt... :!:~. pp :.~l-:\1'

1:1. I.ilh H.\, 1.11\..,,11, F.li. 1l''��II('liClII''1 '~1l1 .\11 ,'''..''11'''111 "1101\.,vn:l'aroiiiClIl'. Hi .\lnllI'I~I. :\:;~4-li~li

14 I.Clwh1i1' ~.. I. I.ilh H.\ îl". ",.. 01 4 '; 1 hl"II"',,,11i1i tI"I('II:"1I1,,011I1I011 (Hihi,cnihl.iiid ocht:r nll"IH)(I" 01 ,kiii di.iiiln 111111. n, \1.,/I 1117:\: 1 :.-, III-~, I ~,.

1;1. 1.",,1,,1, Ell. I.il" fL\. .\dilk (;.\J I'n'''IllI,IIIH tI""II,, """ "i

"iirgt.oii'" h.lilcl"': l...c..iil .i1illhl,lil ~C)llllll)i'" ,IIHIl'IIl' '..iil ~Iii\(', fIll'~1I llii.i. H. .\1,.1 ¡. 11174 4::11;11-:174

lti l.ill- H\. 1."'.Il1l1' Ell. Wil~ii". \Ill. 1'1.01. Ikl,l\l.iI ,1111111111 ",h",i

t'ltt0c.I'"ut ,llidi..ililti'iiIHl In .d(ohl)ljfl\l!.(.flm;IIIj~i¡. ..~.~lj7.-1jii'17 \Iidi.iiicl K'\, \h(.I.1i1i \IL (;"" \\..\ ..\""1'',111''11' "i .. ~I""i!

hand "llidt'1 1111 II111hlp.n.il1ll'lt'r 1I1l.I..un'IIU:llh ill ..liI1\ ~i" Ill\( itil",""!' l ( I", ,\Iii ,,,""'1 i'iïti: :14111,-41:1

II'. Reh.:1 1\, \ltliill'II'1 \1. ,.., ~ 10. \,1.,1 /111 1'1 1i1111('lll"tll~ iki ,i".riii~:.t'.., ht.'11 ll.iiidl,' dl"'Iii1(,i lIIIJII I J~iitf /l,:.' .~h' (III:. Ill~"IÔll:.."I-ii:!~

I 'i il"",,, I, K"hhl K, .\ \ Iiii" (,.\1. ''I .11: Il"iii ki 11"" "i iii.. -~Iii "i 11"ahcl"III'II. Hi l'''i.!. i'i~i-. li~,I":-i.-,,~~,i-.

:,l1, 1.0\\')\11" E.lI..l.i1h HA' (~I()\t~d h~iilll il'" ;lpplic..lllll 01 ,iiiii..t'lilh i.Upt'T,lIiul1,lte:- l.fi'''''' 1~i:L :!:I..):~.i..iti,

:!I. n",.I,".I B..hhlK, .-\ di lit' (..\.1 1'1 "I: Th\' I'kiiiiillli.. ,~iiill"i" "ihaihin~ \\iih aiiii"t',uit 'OlliIIClII....1 ,-\"'IIIIIt ii,i, L/ii'iilil/h, i 1~17-::1:4i:1--l~1

:!:!. Braiidlii'ii: .\, .-\lilkr,,"1I I: \\1111I'-IHI(I- di,,"kn,"" I" ,ii "''' ,

haih wll h ,hl"rh",icliiii' '"av I'rl',,'IiII'd .11 ill( 1'lIi'nl", "'ii,ii ( "".loT''" aiid "i, il IKi-¡1i il "ii Pr"IiI,'Ii1' II iii\' L"Ii11 ,,1111 1i"-,,il,,1 I II I.. .lion. L.undoil. 1~.Ktt. Hii,,¡/ .'"ru''-, ill .\lniln,,, "l"Ilt".. :!:t. pp I~.~l. '"11

:!:\. Crii,l' 1'.1 L ~'H .nl K: .-\ hH'-H'"'' prii,I"'II''' '1111 h III ~:i.104'' '"\~" "ipaiienis .i'''/¡ .\ic.i: I'I~:I: 1117 ::!lIl;-:!IIII.

:!4. Krlili".I'. Po,ioper;ili\( "oiinciiiieuioii. .itlli.\I..! '''/lH/l~I~Il: I:'i~iippl '-11-i:1-~'I,

:!..l. 8r;,lliclhc.'I'~ .-\. HClIIII J Híiiiiinar,it'li J ~l hc.'I"'ll'lI I' PU"IIIPI.'I,III\i.~fllIlid inlt'fiit JllS IIi V.i!' ular lõlllllt'n -~.l It'il III pIC:11pt"l.ii 1\(" ,\ hi 'Ilhoò\ disintt"riioii t1\ ,hii\\i'r.haih \,'jih t hlurlu.',IlIIlIC ....,ip PH.'tllIl"cI ~ii i ht' Illl~rnaiiol1.il ClJn~n'~", and ~\ Illpo"niiii 111\ Pn Ihkl1"in iht. Ciiiiinil of Ho",pililllnlt.("j,1i1' L.ondoii. PI~II. fi.,,¡'l 'i,,,wfì i.~.\1,./,''11,. "i'rit... ~:~. pp ïl.'7:i

:!li. .\\ Iifli' (;.\.1, ''' \1 F, Bahhl R,,,i al: .\ i OIlI',II'i.iil1 III ~11..,,1,...111\1haihiig "ii li ,hlorht,~I1II1I" dl'lnlol'lil .iiid 1i01i-lm,dOl "i,'d '''''I' ",iht, Pll'\t,'1l1l011 nl \\1 HUul iIlICl'(ioli./fJIIIJlII¡ "! 1I,,,I"ltll 1"'11 i,.". I~I."'.)

4::!:\~-:!H.:!7 ~.t"ht'q:~. Kn'llill;11I BR: l~Opt'r.ili\t. lcliL11 \\..,hln~\'llli l hlliriH'"

.diii.. i.1ii\iII..IO' f,,'rlornllcl I" p.llil'nls iindnloiiini: iinlilll',,,'d,,~1I"l(l'l'. Pn..nltci ai ih,' Inll'rli;ilion.11 Loiign'" ,111'1 "i'IiI"""II;lin l'rohkii, iii iho' COnlrol ii( Hospil;11 Inll'llloli, i.iil1i1"Ii. 1'1"".nltWl/ ,\,.."'\ "i ,\/,./"11''. 'l'ril" :!:~, PI' ii .

!til

:!!', lavlor L.I \Ianiii H: Pr~opt'raliH' .kin pllp;ir.iiiiin 01 "nhClp,Ili1..

Jl\ii'ntli, .-\ """il/ eii",,,,,,,,,,, ",/iif,.(i",ii l ;""""/'\,,, ,,- I ~1';7 . Pl' :!~l. .\:::!!I. 1)",.i("oli ,-\1(;, Clarkt" C. Smiih (~: 1\"(01)(1'.111,, '''..111(\ 11111.' 11""

.-\ l.iiiipUll'r aiial"i~, H,./ ,\"'.1: I!lil: ;i,,::iI:,:I:I~.:~II. l\trr ..\R, Waii B, (;olciacT'' M.J 1'1 oil: .-\ (i'lIpOl..i",1i "i 11-" III

1''' id'lllt-iociiil' ,mci dilorht"idiiit' iii ihi' pmph, 1.."1, ot IH "'-"1"'1'allH' wouiici illll'nioll../eliinmi ell H'"plltl/ /11/.''',,'' 1!11':!: :1:;,.-'-1;:1.

:\1. Ml'l'J" PO, .-vlifft" (;.-\.1 Emm..rsoii AM, 1'1 oil: Rqiiirl oil 11i'.'aiioiial Sunt' 01 Inil'nioii iii Hospiial, 1!1!t1l.101"nUli oil /1'''1"'.;1

/"/"(1/111 l!ltll; :!isuppii.:\:! 1..d...t.1I 0\1. LIIWbli1' E.l L. \\hnt" \\ l'l al: .111' l'f1l'CI' 01 lilt r;l-rl,',1I

air iii lIptraiinii roiim, oU dl'l'p sl'psis alil'r lOlal hip or kiltI' rI¡iI.IlI'-mt'lI, Hi ,\I,d J I!li':!: :!II:: 10.14,

:\:. :hliltl' (;A.J Bahh.JR, Brinjtl's h.. ~I al: eollVari,,1i1 1111\'" 1I1'IIiIHl-

10" ¡¡'t"s'lIlllhl' ltlio,,,1 Olltl.al oriiaiiism, ..iici ¡i.iihiigi'ii- Iniiii Ii",,kili..1 H\Jl (,tliih l!li;,: ~'-,:~,'i!l-:!7 4.

:H. Ta\'lor 1..J .-\11 I'\alu"iioii ofhanci'''ashiii¡: Il'chii,!"i" Sii"iiii. rii""1!l7!t: ï4:H-llO.

:\:. ("Hinaiil.i~t" I'LL. :hliflt' (;:\,1 l:ih..ricijtl' KA: F"l1ur' ,'''IKi.It'ciwiih ihi' ri.k 01 i"isiuf,,:raii\'~ .."ullci illll'niuii .111(\ 11..,.11, .11T...g" ul."fiph. tlllll'II', IJnHI'''¡'''K' "iihl' /-Iih H(I;1

'1 II ,''1'.' "I1II11m" l .11",..".", / (If

.\\',1'11 .'ori"., l!l!tl: LI:Ô~I-~(I~.

~;

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I

Journal of Hospital Infection (1988) 11, 226-243

Hand disinfection: a comparison of various agentsin laboratory and ward studies

I

G. A.J. Ayliffe, J. R. Babb, J. G. Davies and H.)I

Hospital Infection Research Laboratory, Dudley Road Hospital, BirminghamB18 TQH and *MRC Burns Research Group, Birmingham Accident Hospital,

Birmingham B 15 iNA

Accepted f01' publication 8 June 1987

Summary: The effcacy of 14 handwashing or disinfectant preparations wascompared in laboratory tests on staff volunteers. The test organism, Escheri.-chia coli, was applied to ihe fingertips and log reductions (LR) were measuredfollowing treatment with the test agent and control preparations (70%isopropanol and non-medicated bar soap), Alcoholic preparations, particu-larly n-propanol and isopropanol were the most effective showing LRs of3'1-3,8. Chlorhexidine (LR 2'9) and povidone-iodine detergent preparationswere significantly more effective than non-medicated soap (LR 2'1), bUL.

- triclosan J'roduc:ts were not. In addition the residual effect of several of these

'formulatIOns was. àssessedafter 10 applications by comparing the survival of

E. coli on the fingertips over a 32-min period. This number of hand washes. c:ompares favourably with those recorded during an 8 h nursing shift.

Chlorhexidine-detergent consistently showed the best residual activity. Alco-holic formulations showed little or no residual effect. The survival studiesshow that on the whole gram-positive organisms (Staphylococcus au

reu andCandida albicans) survive better on the skin than Gram-negative bacilli(GNB). However, it would seem that GNB which are considered to beresidents (Acinetobacter calcoaceticw and Enterobacter spp.) survive muchbetter than many other GNB (Pseudomonas aeruginosa, E. coli and Proteus'Vlgaris). The Klebsiella species varied in survival times. Random samplingof ward staff hands showed that contamination with S. au reus and GNB wasgreater in dermatological and general wards than in an isolation unit, wherehandwashing or disinfection was carried out after every patient contact. Nocross-infection occurred in the isolation ward during periods of study inwhich 70% alcohol, chlorhexidine-detergent and non-medicated soap wereused.

Key words: Handwashing; residual effect of disinfectants; disinfection of hands.

IntroductionHandwashing or disinfection is one of the main methods for reducingcross-infection in hospital wards (Reybrouck, 1986). I ts effectiveness inreducing the spread of organisms depends on several factors; the occasione.g., before carrying. out aseptic techniques or after handling bed pans, thepreparation used, the technique of handwashing and the extent of

OI9S~701'8810J022b+ 18 SOJOO¡Or 14!'8 Thr Hospiial In(tetion SOCIC'I:'

226

oj

(

(

(

c,

'l_

228 G. A.J. Aylife et al.

Two minutes after the application of test organisms, the fingers and:thumbs were immersed in a bowl of 100 ml of nutrient broth (Oxoid No 2),j

containing neutralizers appropriate to the disinfectants used, and rubbedvigorously over glass beads (3-5 ro diameter) for 1 min. Using a surface ,

dropping technique. 2 x O' 5 ml of the neat broth and 5 x 0.02 ml of tenfolddilutions were transferred to the surface of well dried blood agar plates and -incubated for 18 h at 37°C. Surviving test organisms were counted to enablecomparisons to be made between individuals and products used.

Test bacteria were reapplied to the fingertips and after 2 min. thestandard formulation, i.e., bar soap or 5 ml of isopropyl alcohol. was appliedto the hands using a standard technique compromising of five strokesbackwards and forwards; palm to palm. right palm over left dorsum, Ieft-palm over right dorsum. palm to palm with fingers interlaced, backs offingers to opposing palm with fingers interlocked, rotational rubbing ofright thumb clasped in left palm and left thumb clasped in right palm,rotational rubbing with clasped fingers of right hand in palm of left handand clasped fingers of left hand in palm of right hand. The hands and wristswere rubbed in this way until the end of the 30 s period. Finally, the handswere either sluiced under running water and dried on two paper towels(soaps and detergents) or alÏowed-tor dry (alêöhorr for a further 30 s.Surviving bacteria were recovered in the manner already described.

The whole procedure was repeated using the product under test and themean logio reduction in test bacteria calculated using the proceduresummarised in Figure 1. Wherever possible, the manufactuers'recommendations were followed as to the quantity of the product applied,i.e., approximately 5 ml of liquid soaps and alcohol, 2.5 g of foams andsuffcient bar soap to obtain a good lather.

The products tested included the following alcoholic formulations: 70%isopropanol or 70% ethanol with 1 % glycerol as an emollient (standard),'Sterisol, (Seton Products)-60% n-propanol, 0.5% isopropyl myristateand 1.5% glycerol; 'Sumagel, (Lever Industrial)-48'5% isopropanol,gelling agents and emollient; 'Hibisol, (ICI PharmaceuticalDivision)-0'5% chlorhexidine gluconate in 70% isopropanol and

íAP01Y organismi TronSPose piote

I lleco~e,'orQonlsm-coISIS 10 10910 -Meon iog pre-alslnltCfloniI 5ysle",10-15 lesl persons I..iin .."'IOC.I skin, :iCPlY J I I SIOr"oorCl ::ra ies!firs' Ihe stondard ona Re.opply crQonlsm I Cclcul:::I'e mfor. 'oq reou.'. ':0l"IP'en 'I'e itSI I _ 'ocror IRF 110:; pre-Oisi".ec~:orip~eDorOlion _! I -iog POSI-ois,n1eci.or."OQ RF'Appiy V.lf. ¡ .

Ol$in,..cioni 1 AODI)' " It'S! 10 meo,. 'oq íl~s 01or OICOI'Ol I 'esi end s'O!"-Ooro D'epO'C!lol'.5,(5,10noorO 0' 115011 J .Tron~00501 plOl1

RecOvel oIQonl50m-Counl!i 10 loq,O -Meal" IOQ PCX1-0150lnleClionsystem

Figure 1. Hygienic hand disinfectant procedure: calculation of effcacy of test and standardskin disinfectants.

i I. ~ .

I:. . Hand disinfecûon studies 229-~~ollents; 'Manusept', (Hough, Hoseason & Co. Ltd)-0'5% triclosan in.". 0% isopropanol and emollents; 'Hexifoam', (Stanlee Australia)- i %

~cbiorhexi~ine gluconate in 60% ethanol with propell~nts; 'Alcare', (Vestalbt.Labora.toneS)-62% foamed ethanol; and the followi?g detergent scru~s:~~Unedicated tablet soap Type B (standard). (N imbus Laboratones

~".Ltd)-conforming to BS 1914 National Health Service supplies code MRA'.~560; 'Hibiscru~:I_.(lCI Pharmaceutical Division)-4% chlorhexidine;"'t~giucona~ clean~er; 'Betadine'. (Napp Laborat~ries Ltd)-7'5%. 7-povidone-iodine surgical scrub; 'Gamophen', (Surgikos Ltd)- i ,5%

.'trclosan antiseptic tablet soap; 'Zalclense' , (Sterling Hospital

. Products) 2% triclosan bactericidal wašl.cr; 'Aquasept', (Hough.oseasn & Co. Ltd)-2% triclosan skin cleanser.

. '. Neutralizers were inèorporated ¡; th;-n-;~~;r- broth used tò recover.- iving test organisms and the same medium was used for standard and

le fòrrulations. Neutralizers were as follows: 0.75% lecithin-Tween

Øire(SO g Tween 80 and 5 g lecithin) for chlorhexidine containing~ormu18tions,i % sodium thiosulphate for povidone-iodine formulations_ind3%Tween~0 and i % lecithin for triclosan containing formulations.-.,

urval 0 transient 0 nisms onnonnal skin

e survival of specific organisms on the skin was investigated by. . JÎioculating the fingertips of each hand with suspensions and periodically". sapling for survivors (Ayliffe et al., 1978; Casewell & Desai, 1983).

~~, Twenty-four hour broth cultures of specific micro-organisms. isolated. rrom wounds and urine, were washed twice in Ringer's solution and

- uspended in 10 ml of distiled water. One drop (0'02 ml) of the prepared_ _ suspensions, was applied to the palmar surface of the fingertips of

,.¥olunteers who had previously washed their hands using unmedicated bar"::fsop and who had not handled disinfectants during the preceding 48 h.'::;,Opposing fingertips of the left and right hands were rubbed together for 40 s.:;no disperse the test organism over the palmar surface of the finger tips which'..i.!_ere left to air dry for a further 80 s. Fingers were then sampled at specific..:~: tie intervals. i.e.. 2. 4, 8, 16, 30, 60, 90, 120 and 150 min after inoculation?-: by immersing them in 60 ml specimen containers (Sterilin, 125 BM)'~i:containing 10 ml of saline broth (i.~.. normal saline plus 10% nutrient':-:l.broth) and rubbing them vigorously over glass beads (3-5 mm diameter) fortJ min. Two fingers, one from each hand, were sampled at each time interval.:;;~d the samples pooled. Ten volunteers were used for each test organism~;.and sampled on two occasions, i.e., at 2, 4, 8, 16 and 30 min and 2, 60, 90,:~120 and 150 min.

,~Tenfold dilutions were prepared from the washings at each interval, and:,;'using an appropriate enriched medium. surviving test organisms wereoffrecovered by a surface dropping technique.

.'t The mean logarithmic reductions of test organisms at each time interval,...~2 "ere calculated and the results plotted as curves for species comparison.

- ..- -

;,-' ,: ;~-l(

j

I

i

'I

i

23 G. A. J. Aylie et al.

Test organisms were as follows. From bums wounds: Acìnetobacter't;:calcoaceticu var. anitratus, Candida albicans, Serratia marcescens,:.i

Pseudomonas aerginosa, Proteu vulgaris, Escherichia coli, from an infected.lpostoperative wound, Klebsiella aerogenes and from infected urine, K. -!t-aerogenes serotype 21. -t. ,.---- "" .~=i~~:lth:ti:::l¿al or persistent activity of skin disinfecttÎs, voiunteers-l

.. 'nand short finger nails washed their hands 10 times during a ~

6 h period usi g the five stroke technique described in the previous section.-'JTen . s after the final application of the formulation under test, the ~ ipalmar surfaces of the finger tips of both hands were inoculated with 0.02 ml ).1of an overnight broth culture of E. coli ATCC 11229 and opposing fingers -':land thumbs rubbed together to disperse the organisms over the palmar :Ls~.r~of the riger ti~s of 40.s. At specific time intervals, i.e., 2, 4, 8,.16 and ~: ¡-32 mm after moculatlOn, paired fingers/thumbs were sampled usmg the. ¡

__~thôds previously described. An appropriate neutralizer was incorporated .: iinto the recovery broth.

Surviving test bacteria were enumerated and the mean loglO reduction of :: itest organisms calculated at each time intervaL. Formulations tested were as j,-,follows:- unedicated tablet soap, 'Betadine', 'Hibiscrub', 'Zalclense',;;-'Aquasept'. 'Gamophen', 'Hibisol', 'Manusept' and 70% isopropyl alcohoL:),Another control was included, in which all the volunteers had no treatment Lbefore inoculation of finger tips. Wherever possible, the same volunteers ,~¡

were used to test each formulation and care taken to exclude volunteers-'~:routinely using antiseptics or disinfectants. ..

~',.

Transient hand carriage in general and skin hospitalsThe hands of nursing staff in the wards and isolation unit of a large districtgeneral hospital and a specialist skin hospital were sampled for transient 'carriage of S. aureu and GNB. Unexpected visits were made to the wards at.different times and the nurses' hands were sampled without prior washingor disinfection. Hands were sampled by rinsing them in bowls of sterilenutrient broth (100 ml) containing antiseptic neutralizers, i.e., 0.75%lecithin- Tween mixture (50 g Tween 80 and 5 g lecithin) and 1 % sodiumthiosulphate. A fivefold rinse technique was used (Lowbury & Lilly, 1960;Ayliffe et al., 1978) and approximately 10 ml of the sample removed for -culture.

Within 1 h of sampling, tenfold dilutions of washings were enumerated.Two recovery media were used; blood agar plates for total counts and GNB,and phenolphthalein disodium phosphate agar for S. aureus. Plates wereincubated for 18 h at 37°C and transient flora enumerated. Gram-negativebacilli were identified using Analytical Profile Index System (API) and S.au reus confirmed using the deoxyribonuclease (DNase) test.

Unmedicated bar soap was used for handwashing in all wards with the

"~ c~

Hand diinection studies

~ception of the isolation unit where bar soap, chlorhexidine detergent and(.ii.,,.aIcohol were used. A record was kept of each visit, i.e., date, ward, products

)Fused, nurse sampled, and activities undertaken.~¿:. Electronic handwash monitors were installed in a male surgical ward to

iGstudy handwash frequency of nursing staff. The monitors (Broughall et al.,';'1984) recorded a single handwash provided the member of staff stood in~'front of the sink, applied one or more aliquots of liquid soap from the

( .....dispenser and removed one or more paper towels..~~ .

""Transent hand carriage in an isolation unit-the effect of antiseptics~The isolation unit is used for source and protective isolation of. predomiantly adult patients (Ayliffe et al., i 979). These include the,uiunologically compromised, carriers and dispersers of multi-resistantstns of S. aureu and patients with tuberculosis, gastroenteritis and otherCommuncabJe diseases. The ward consists of single-bedded cubicles, eachWith its own hand wash basin.'~Usingthe inethod outlined in the previous section, the hands of nursingsta were sampled, on a regular basis, over a 45-month period.-~. Handwash formulations used were as follows: 4% chlorhexidinegluconate skin cleanser ('Hibiscrub'), 11 months; placebo, i.e., 'Hibiscrub'Without chlorhexidine, i month; 'Hibiscrub', 6 months; unmedicated bar';aap, 23 months, and 70% ethanol (with the addition of i % glycerine as anéiollent), 4 months. With the exception of 70% ethanol, the products were

us exclusively during the periods indicated. As alcohol cannot be used ford cleansing, bar soap was often used prior to disinfection.

(

ResultsHygienic hand disinfection

.lThe effcacy of various soaps, detergents and alcoholic hand rubs is shown

:in Tables la, Ib and 11. The antimicrobial effcacy of each formulation is.expressed as the mean logarithmic reduction in test organisms. A mean log:reuction factor (logio RF) of 2 is equivalent to a 99% reduction in test~organisms, a log RF of 3 to 99'9%, and so on. Tables la arid Ib compare the:effcacy of each product tested with that of the standard formulations, i.e.,either 70% alcohol or unmedicated tablet soap, whereas Table II expressesJhe effcacy of formulations in their rank order of effectiveness. Statistical'Cmparisons were made using the student 't' test between paired samples(i.e., test and standard) in Tables la and Ib (Swinscow, 1976a) and unpaired~- . pIes in Table 11. (Swinscow, 1976b). Differences are considered.gnificant if Pc: 0.05.. In paired tests (Table Ia and Ib) chlorhexidine gluconate skin cleanser~Hibiscrub') and povidone-iodine scrub ('Betadine') were significantlymore effective than the standard unmedicated tablet soap. Triclosan()rmulations ('Zalclènse', . Aquasept', and 'Gamophen ') were not.

231

I'

232 G. A.J. Aylife et a1.

Table la. Hygienu hand-disnfection test: statistical comparison of diference between means oj;paired samples (test an standard) . - .-i:

Mean No. oflog observations Significance -~

Soaps and detergents RF (tests) Poe 0'05 :s.);

# 'Hibiscrub'

~4% Chlorhexidine gluconate 2.9 20(2) S

=1'Unmedicated tablet soap 2.2 Poe 0.001

# 'Betadine'

'~l7.5'1. Povidone-iodine surgical scrub 2- 45(4) S c_

=j,· Soap 2- Poe 0'001 '"

# 'Zalclense'-,"

2% Triclosa washing crea 2.3 20(2) NS ,'j·

Sop 2.0 Poe 0.1 ::0.05 ".. ~

# 'Aquaspt ..'

2% Triclosan skin cleanser 2.3 NS_1

7(1 ) ~.i· Soap H PoeO.5::0.1 -":1

# 'Garophen' .'.~

1.50/. Triclosan tablet soap 2.211 (1)

NS'Soap 2.1 Poe 0.5 ::0.1 -

# test; · standard.S, Difference significant; NS. difference not significant_ oj,

~"...:-lJi

Table lb. Hygienc hand disnfection test: statistical compaTison of difference between means of .~paired samples (test and standaTd) '!:.~.:~

Alcohols

MeanlogRF

SignificancePoe 0,05

c;~j;...':!;f

No. ofobservations

(tests)

# 'Sterisol

60% n-propanol gel'70% Isopropanol# 'Sumagel48'5% Isopropanol gel'70% Isopropanol# 'Hibisol0'5% Chlorhexidine gluconate in 70%isopropanol70% Ethanol# 'ManuseptO' 5 % Triclosan in 70% isopropanol# 'Hexifoam'1 % Chlorhexidine gluconate in 60%ethanol foam'70% Ethanol# 'Alcare'62% Foamed ethanol'70% Ethanol

J-10(1 )3.5

H10(1 )H

3'1 24(2)2.7

3.1 28(2)2.9

2-10(1 )2.6

2-5l2( 1)2.9

NSPoe 0.5:: 0.1

NSP::0'5

SPoe 0.05:: 0.02 :,

NSPoeO.5::0.l

NSP;:0-5

SPoe 0-05 ;: 0-02

,# test; . standard.S, Difference significant; SS. difference not significant.

B10

.~

9

'Ste

riso

l'60

% n

-pro

pano

l gel

'Sum

agel

'48

.5%

Isop

ropa

nol

gel

(Sta

ndar

d)70

% Is

opro

pano

l'H

ibis

ol'

0.5%

Chl

orhe

xidi

negl

ucon

ate

in 7

0%Is

opro

pano

l'M

anus

ept

O' 5

% T

riclo

san

in70

% Is

opro

pano

l'H

ibis

crub

'4%

Chl

orhe

xidi

negl

ucon

ate

(Sta

ndar

d)70

% E

than

ol'H

exif

oam

'1

% C

hlor

hexi

dine

gluc

onat

e in

60%

etha

nol f

oam

'Bet

adin

e'7-

5%Po

vido

ne-i

odin

e'A

lcar

e'62

% E

than

ol fo

am'A

quas

ept

2% T

riclo

san

skin

clea

nser

'Zal

clen

se'

2% T

riclo

san

was

hing

cre

am'G

amop

hen'

\'5% Tríclosan

tabl

et s

oap

(Sta

ndar

d)U

nmed

ícat

ed ta

blet

soap

'"

3.4

NS

C D

89 242 3 2 5 4 2 \3

3.3

3.\

NS NS

S NS NS

E28

3.\

S NS NS NS

== ~ Co e: l It n go.

o ~ ~

,.-a.

......

.je:

" ..

ii:

F20

2.9

S NS NS NS NS

G H

56 \0

2'7

2'6

S NS S NS NS NS

S NS S NS NS NS NS

46

J12 7

2.6

S S S S S S NS NS

K L20

2.5

2.3

S S S S NS NS NS NS NS

S S S S S S NS NS NS NS

M\\

2.3

S S S S S S NS NS NS NS NS

N\3

5

2.2

S S S S S S NS NS NS NS NS NS

2.\

~ wS S S S S S S NS S NS NS NS NS

23 G. A.J. Aylife et aI.

With the exception of 70% isopropanol with O' 5 % chlorhexidine;. ~('Hibisol'), none of the alcoholic formulations was more effective than the'Jstandard formulations, i.e., either 70% ethanol or isopropanol. Isopropanol-~was considerably more effective than ethanol and this probably accountedt:for the significant difference between 'Hibisol' and 70% ethanoL. '~:'

The most effective formulations (Table II) are those containing alcohoL. ~'These give logio RFs of

between 2.5 and 3.8. Formulations containing either..(-n-propa~ol or isopropanol were stastically more effective than those ':i~containing an equivalent concentration of ethanol. Alcoholic solutions and ~¡

gels were more effective than foamed alcohoL. The addition of 0'5%-~.

chlorhexidine or 0.5% triclosan did not improve the immediate effect of the ~alcoholic hand rubs.-On the whole, the soap and detergent scrub formulations were inferior to -

those containing alcohol, although all were significantly more effective than.unedicated tablet soap with the exception of those containing triclosan('Zalclense', 'Aquasept' and 'Gamophen'). Chlorhexidine gluconate ski.cleanser ('Hibiscrub') was notably more effective than other soaps anddetergents and was not significantly different from many of the alcoholichandrubs. It was also significantly more effective than povidone-iodinescrub (Betadine), triclosan containing products and unmedicated tablet.soap. The three triclosan formulations were significantly no more effective:~than unmedicated tablet soap. -

..

oAcmetobocter colcoocetlclJS

Condido olbiäonsi

\ . '..... - - - - - -\ . -~-------------\ '. __ StophylOCOCCIJS OlJrelJsI .-iX,.b~~ "iI ~16'0 ¡--\ . __ -- x.,\ ----0, .--x. ',serrotio ~

'- '- ,morcescens'- "-..__ , x- -" _ _. PselJdomonos '\

, oerlJçlnoso '\'- X EscnenchlOProtelJs VlJlçorlS ° coil

--- ---- ----

-.i:eü::

'0~

~ zlo-'

3~

I

21

16

1

30I

60i

12090

Time lmin)

Figure 2. Survival of micro-organisms on normal skin.

Hand disinfection studiesÚiiival of transient organisms on normal skin

-' e reduction of specific micro-organisms applied to normal skin is shown11' Figure 2. As the reduct!on ~ver the the sampli?g ~nterval is rap~d, t~e:; ults are expressed logarithmically. The curves indicate the logarithmic

.. uction in test organisms at specific time intervals over a 150 min sample-':od..Gram-positive organisms, i.e., S. au reu and C. albicans, survived

plcularly well on the h.ands wit~ little ~ore than 1 log ~90%) re~uction ~numbers over the 150 min sampling period. Much of this reduction was in.~ initial 2 min drying period. Gram-negative bacili varied in their., ponse. Acínetobacter calcoaceticu survived more readily than any otherra-negative test organisms. Serratia marcescen, P. aerginosa, P.-ar and E. coli survived poorly. Klebsiella aerogenes was more tolerant,

ülôugh test strains varied.

~ --.:. ual activity.- e loss of viability of transient organisms applied to the finger tips after'-applications of each product is shown in Table III. The greater theuction at the'same time interval, the greater the residual or persistent

èc. The reductions are again expressed logarithmically and the productsië in ranking order of effectiveness. Statistical comparisons were made~e~n all the products listed using the students 't' test (paired samples).iferences were considered significant if P -e 0.05.ëhlorhexidine luconate skin ~.a&e.I_CHi1?t~crub~) shwedmarked'du activity and Was significantly and consistently more effective than

~'õther products tested. Povidone-iodine scrub ('Betadine') was

.¡icantly more effective than bar soap, but not significantly better than'.rtriclosan containing products 'Zalclense', 'Aquasept' and 'Gamophen'.. -e alcoholic forrulations('Hibisol', 'Manusept') with or without the

~ádition of chlorhexidine or triclosan showed no significant residual effect.~~'

,~

rcment hand carriage in general and skin hospitals~rasient hand carriage with S. au reus and GNB is shown in Table iv. Of.èd nurses' hands sampled on surgical, medical, gynaecological andd hopaedic wards of a general hospital, 29% yielded S. aureus and 29%NB. The incidence of hand carriage with S. aureus was, however, much¡her (78%) in nurses working in the wards of a skin hospital whereá~ients with skin diseases, such as eczema and psoriasis, were being nursed..~antibiograms and phage types of these strains varied with the sampling

_;'and occasionally several strains were isolated from individual nurses.ere Was no evidence of persistent hand carriage with a particular strain of

-' Clieu. Antiseptic handwash formulations were rarely used in generald skin wards and the skin hospital nurses preferred not to wear gloves.

;.On the isolation unit, transient hand carriage was subtantially less than on"."er wards, i.e. S. au reus 14.9% and GNB 16.7%. Antiseptic formulations,

235

.~. ~~.~".:.

! '.:-fli~r

.t~;f~~::d:~Zft::

';.' ,'-'.". ~

;~i~.:

Table III. Residual activity following 10 applications of formulations under test (10 volunteers per treatment)

Sam

ple

inte

rval

2min

4min

8min

16m

in32

min

Mea

nFo

rmul

atio

nR

FR

OR

FR

OR

FR

OR

FR

OR

FR

OR

O'H

ibis

crub

'4%

Chl

orhe

xidi

ne g

luco

nate

2.80

13.

251

4.20

14.

641

4.92

11

Ç)

'Het

adin

e'

~7.

5%

Pov

idon

e-io

dine

2.66

23.

062

3.49

33'

455

4.37

42

..'Z

alcl

ense

'

;i2%

Tri

clos

an w

ashi

ng c

ream

2.07

.4

2.58

63.

542

3.94

24'

493

3~

'Aqu

asep

t'

ri2%

Tri

clos

an s

kin

clea

nser

1.73

72.

694

3.00

63'

763

4.86

24

~'G

amop

hen'

II1.

5% T

ricl

osan

tabl

et s

oap

1'72

8.2

.78

33.

484

3.71

44.

071

55

~ liU

nmed

icat

ed ta

blet

soa

p2.

005

2.64

53.

205

3-2

63.

n6

6~

'Hib

isol

'0-

5% C

hlor

hexi

dine

in 7

0% I

sopr

opan

ol2.

383

2.51

72.

987

3.20

73.

279

7'M

anus

ept

0.5%

Tri

clos

an in

70%

Iso

prop

anol

1.74

62-

"10

82-

09

3-3

93'

408

8N

o tr

eatm

ent

-1'6

99

,.2.0

99

_ 2.

828

3-0

8. )

,64

79

70%

Isop

ropa

nol

1-68

102.

0010

2.41

102.

6910

3.15

1010

RF,

Mea

n I(

)~ r

educ

iion

in le

st b

acte

ria;

RO

, Ran

king

ord

er o

f ef

fect

iven

ess.

~

..

Table iv. Transient hand carriage

:: I)

Staphylococcus aureus

Gra

m-n

egat

ive

baci

lli=

' Q.

% S

how

ing

Med

ian

coun

t%

Sho

win

gM

edia

n co

unt

e: II

No.

of

sam

ples

grow

th(m

axim

um c

ount

)gr

owth

(max

imum

cou

nt)

e: It

396

14.9

10,0

016

.738

,000

n ..

(980

,000

)(i

3,0

00,0

00)

õ. ='

4678

.338

,00

30.4

10,0

00II

(14,

300,

00)

(3,0

00,0

00)

a18

029

-43,

800

29.4

3,40

0e:

(24,

00,0

0)(1

2,50

0,00

0)ii

Isol

atio

n un

it

Skin

hos

pita

l

Gen

eral

hos

pita

l

~

:1,'.

Ran

ge o

f cf

u.pe

r sa

mpl

e

/1,0

00,0

0010

,001

-1,0

00,0

0020

0-10

,000

200

(no

grow

th)

Nurses sampled

r'.'"

("""

'"~,

Tab

le V

. Inf

uenc

e of

soa

p an

d an

tisep

tics

on th

e tr

ànsi

ent h

and

carr

iage

of

nurs

es in

an

isol

atio

n w

ard

Perc

enta

ge o

f sa

mpl

es

Hib

iscr

ubPl

aceb

oB

ar s

oap

Alc

:oho

lP

Stap

hylo

cocc

us a

ureu

sG

NB

S. aureus

GN

BS. aureus

GN

BS. aureus

GN

B?" ': ;¡ -.

°°

°0

01.

80

0fi

5.0

10.1

6.7

6.7

3.7

11.9

7,4

1.5

~11

.910

.71.

73.

38.

39.

213

21.

5" ~

830

79.2

91.7

90.0

88.1

77.1

79.4

97.1

~15

960

109

68

.col

ony

form

ing-

uni

ts.

£j 00

~.

~.:~

/..

(

(-

240 G. A.J. Aylife et a/.

The effectiveness of the antispetic detergents and foam varied but were.generally more effective than unmedicated tablet soap. Those containingeither chlorhexidine or povidone-iodine were the most effective. There wasno evidence that triclosan was more effective than the other preparationstested (Bartzokas et al., 1983) against transient organisms. Although theagents are placed in order of effectiveness, the differences are often smalland insignificant. In some experiments only 10 volunteers were used.Nevertheless, all of the experiments were well controlled and the results ofthe paired samples were usually similar to the overall results. In the.interpretation of the results it must also be remembered that E. coli was the.test organism and results could differ with other organisms such as S.aUTeu, although the available evidence suggests the differences will not begreat (Ayliffe & Babb, 1979). The time of washing may also be relevant. Insome laboratory studies applications for 1 min are used; in this study a 30 sapplication was used but, in practice, 1~20 s might be more realistic(Taylor, 1978a).

The survival studies confirm that S. aUTeus survives well on the skin and

most GNB survive poorly (Lowbury, 1969). There are exceptions: A.caicoaceticus vaT anitTatus and some epidemic klebsiella strains show bettersurvival, increasing the opportunities for spread on the hands (Casewell &Phillips, 1977). The method of deposition and the suspending medium willinfluence survival (Lily & Lowbury, 1978). Some G NB are encountered soregularly on the hands that they are regarded as residents. They includeEnteTobacter spp. (Hoffman et ai., 1985) and A. calcoaceticus vaT anitratus.Survival may also be influenced by the residual activity of antispetics(Lowbury, Lily & Ayliffe 1974). In this study, most of the antisepticdetergents showed a residual effect and chlorhexidine gluconate

('Hibiscrub') consistently showed the greatest effect. The organism usedwas E. coli which, although convenient, dies rapidly on drying, and it maybe that the residual effect would be mC're impressive against S. au reus.

Surprisingly, the alcoholic solutions showed little or no residual effect.They contain lower antiseptic concentrations, but, unlike aqueousformulations, residues are not rinsed off. Presumably, a larger amount of theantiseptic in the detergent preparations binds to the epithelial cells and thisis not removed during rinsing and drying.

The studies on hand carriage of S. aUTeus and GNB confirm and extendprevious studies (Ayliffe et ai., 1975). The highest carriage rates were indermatological wards where environmental contamination is considerableand handwashing after certain procedures will only have a limited effect.Similar results were found in a burns unit (Lowbury & Lilly 1960; Fujita,Lilly & Ayliffe, 1982). The general wards showed a lower level ofcontamination and 70% of the hands examined showed no S. aureus orGNB. However,some counts were high and indicate a need for moreselective hand washing. The counts of transients in the isolation ward weremuch lower, suggesting a more effective hand washing regime. This was,

indeed, the case since all patients were in single cubicles and staff always

.,¥,

r:~~ Hand disinfection studies 2414,

t _ Wßhed their hands before and after handling patients or their environment.~~:' The agent used had little influence on the counts, apart from possibly the~. use of 70% alcohol on GNB. However, a true comparison is diffcult, since~. the study was not well controlled and the microbial challenge varied in the

t- '. peiods studied. Nevertheless, dispersers of antibiotic resistant S. aureus

l' and carriers of antibiotic resistant GNB and salmonella were present atsome time during each test period. Patients and staff were regularlysapled, e.g., nose, mouth, lesions (where available) and faeces ifappropriate, and there was no evidence of any cross-infection during the

~;- srudy.,:.," The choice of agents for handwashing or disinfection is diffcult since,di:controlled clinical trials involving infection rates are almost impossible to~:( se up except on a very large scale and laboratory assessment remains the

'J.~.:.~.l'... o?ly. availabl~ criterion. The rele~ance of .statistical sig?ificance to clinical

.~:. significance is therefore uncertain and it seems unlikely that one log:~~, dierence in activity would influence the spread of infection in most~":::~ intaces. However, following heavy contamnation of the hands, e.g., if, ~-' contaminated with 106 organisms or more, an agent giving a 3-4 log

.i:;i reuction might be preferred. This would usually be provided by ant. ;.IT., a1oholi~ solution although,. in some instances, an antiseP.tic detergent or1.':;, foa might be preferred. SIXty to seventy per cent n or isopropanol was

í"~' more effective than ethanol in the test and would generally be preferred,

~ :'£ although isopropanol is less effective against some viruses (Kurt, Lee &. ,:~" Pársons, 1980; Tyler & Ayliffe, 1987). The other issue to be considered is

.'. th role of the residual effect in the ward situation. The studies described~~i deonstrate a statistically significant lower difference in survival between~~: hands washed a number of times with an antiseptic detergent than controls.~: Itwould seem unlikely that this difference would influence the spread of~'.. inection, particularly if the hands were contaminated with a large number~.. of ' organisms, e.g., the difference between the most effective antiseptic andi;. no treatment at 2 min was log 1.1 and nearly 1.26 at 30 min, but it is possible

it a greater effect might be obtained with S. aureu. This wil be reported in a1ž~- later publication. The handwash monitors recorded a very low incidence of~. handwash frequency on a surgical ward, i.e., 5-8 per shift. The use offl;. forulations with a marked residual activity may, to some extent,Ii couteract this effect but any suggestion that staff wash their hands less~~. frequently should be discouraged. One of the triclosan formulations, i.e.,:'~. 'Zálcl~nse:, ~as particularly aggressive to the skin. If such. formulations are,~' used in clinical areas, handwash frequency would be considerably reduced.~.. This would have more. impact on increased infection ris. k than the use of~rnre effcacious formulations would have to reduce infection risk.(- In summary, it would seem that the choice of agent is usually

1. unimportant provided it is correctly applied, and at the appropriate time.¡.. '....'. Which particular proced~res resul~ in heavy contaminatioi: have so far not

'; . clearly been defined, but in some circumstances, a more active agent, or one

"+--

.1:,

242 G. A. J. Aylie et aI.

showing a more persistent effect may be preferred, e.g., in intensive careunits or during outbreaks of infection. For most ward proceduresunedicated soap or detergent would suffce but it is important that theformulation is non aggressive and pleasant to use. Unpopular formulations . ~would reduce handwash frequency. ,.

;,~

References...-:

".-l~.~

Albert, R. K. & Condie, F. (1981). Handwuhing patterns in medical intensive care units.NeT England J01rnal of Mediciru 30, 1465-1466.

Ayliffe, G. A. J. (1980). The effect of antibacterial agents on the flora of the skin. Review'';article. J01mal of Hospital InfectiOt 1, 111-124.

Ayliffe, G. A. J. & Babb, J. R. (1979). Hygienic hand disinfection-selection of testorganisrr. Hospital- Hygiene September, 37-41.

Aylifie, G.A.J., Babb, J.R., Bridges, K., Lily, H.A., Lowbury, E.J.L., Varney, J. &Wilki, M. D. (1975). Comparison of two methods for assessing the removal of totalorganisms and pathogens from the skin. Joumal of Hygiene, Cambridge 75, 259-274.

Ayliffe. G. A. J., Babb, J. R. & Lilly, H. A. (1981). Tests for hand disinfection. InDisnfutants: their use and tvaluation of effectiveness (SAB Technical series no. 16)pp.37--.

Ayliffe, G. A. J., Babb, J. R. & Quoraishi, A. H. (1978). A test for hygienic hand disinfection.Journal of Clinical Pathology 31, 923-929.

Ayliffe, G. A. J., Babb, J. R., Taylor, L. J. & Wise, R. (1979). A unit for source andprotective isolation in a general hospitaL. British Medical J01rna12, 461-45.

Bartokas, C. A., Gibson, M. F., Graham, R. & Pinder, D. C. (1983). A comparison oftriclosan and chlorhexidine preparations with 60% isopropyl alcohol for hygienic handdisinfection. J01mal of Hospital InfectiOt 4, 245-255. .

Bradley, C., Babb, J. R., Davies, J. G. & Ayliffe, G. A. J. (1986). Taking precautions: Howurinary tract infection in catheterised patients can be prevented. Nursing Times.J01rTuil of Infution Control Nursing Supplement 31, 70-73.

Broughall, J. M., Marshman, C.. Jackson, B. & Bird, P. (1984). An automatic monitoringsystem for measuring handwashing frequency in hospital wards. Journal of HospitalInfectuin 5, 447-453.

Casewell, M. W. & Desai, N. (1983). Survival of multiply-resistant Klebsiella aerogenes andother Gram-negative bacili on finger-tips. Journal of Hospital Infection 4, 350-360.

Casewell, M. W. & Philips, 1. (1977). Hands as route of transmission of Klebsiella species.British .\1 edical Journal 2, 1315-1317.

Fujita, K., Lily, H. A. & Ayliffe. G. A. J. (1982). Spread of resistant Gram-negative bacilli

in a bums unit. J01rnal of Hospital Infution 3,29-37.

Hoffman, P. N., Cooke. E. M., McCarvile, M. R. & Emmerson, A. M. (1985).Microorganisms isolated from skin under wedding rings worn by hospital staff. BritishMedical Journo1290, 206-207.

Kurtz, J. B., Lee, T. W. & Parsons, A. J. (1980). The action of alcohols on rotavirus,astrovirus and enterovirus. Journal of Hospital Infection 1, 321-325.

Larson, E. (1984). Effect of hand washing agent. handwashing frequency and c1inic:al area onhand flora. American Journal of Infection Control 12, 76-82.

Lilly, H. A. & Lowbury, E. J. L. (1978). Transient skin flora: their removal by cleansing ordisinfection in relation to their mode of deposition. Journal of Clinical Pathology 31,919-922.

Lilly, H. A. & Lowbury, E. J. L. (1979). Limits to progressive reduction of resident skinbacteria by disinfection. Journal of Clinical Pathology 32, 382-385.

Lilly, H. A., Lowbury, E. J. L. & Wilkins, M. D. (1979). Detergents compared with eachother and with antiseptics as skin 'degerming' agents. Journal of Hygiene, Cambridge 82,89-93.

Lowbury, E. J. L. (1969). Gram-negative bacilli on the skin. British Journal of Dermatology81 (Suppl 1) 55--1.

Lowbury, E. J. L. & Lilly, H. A. (1960). Disinfection of the hands of surgeons and nurses.British .Wedieal Journal 1, 1445-1450.

.~

~.::

'.

..~

4.\'..J.

lt,

~...~,..

,!

,:.

-. -'~.-ij

..

Hand disinfection studies243

.LOwbury! E.1- L. & Lily, H. A. (l97~). !J.se of, 4% c~l.orhexid~ne detergent solution;:'. (Hibisrub) and o~her methods of sk.in disinfection. British Medical:lourn,!l. 1, 51~51 5.~:Lowbury, EI L., Lilly. H. A. & ~yliffe.. G. A. J. (1974). Preoperative .disinfectlo~ .of,-., :. surgeons hands: Use of alcoholic solutions and effects of gloves on skin flora. BrItishr": Medical Journal IV, 369-372.

;'~-~Mackintosh. C: A. ~. Hoffman, ~. N. (1984). An exte~ded model for transfer ~f~~, microorganisms via the hands: differences between organisms and the effect of alcoholic-'. ~.~:~ disinfec:tion. JQUrnal of Hygiene, Cambridge 92, 345-355.~'Reybrouc:~, G. (19.86). Handwashing and hand disinfection. Review article. Journal of

..',; Hospital Infection 8, 5-23.

-:Rotter, M., Koller, W. & Kundi, M. (1977). Eignung dreir alkohole fur eine standard'..' -Desinfektionsmethode in der Wertbestimmung von Verfahren fur die Hygienische

;;... Handedesinfektion. Zentralblatt fur Bakteriologie und Hygiene. 1. Abteilung, Orginale

~_': ReiM B 164, 428438.rRotter, M. L., Koller, W., Wewalka, G., Werner, H. P., Ayliffe, G. A. J. & Babb, J. R.~i' (1986). Evaluation of procedures for hygienic hand-disinfection: controlled parallel

:~, .. experiments on the Vienna test modeL. Journal of Hygiene, Cambridge 96, 27-37.,,-S~8COw, T. D. V. (1976a) t tests. Difference between means of paired samples. InJ;r Statistia at Sque One. pp. 3842. Mendip Pres, Bath.

'Swinow, T. D. V. (1976b) ttests. Difference between means of two samples. In Staiiticsat

f... Sqie One. pp. 36-38, Mendip Press, Bath."Taylor. L. J. (1978a). An evaluation of handwashing techniques 1. Nursing Times 74, 54-55.

!--Taylor, L. J. (I 978b). An evaluation of handwashing techniques 2. Nursing Times 74,~': 108-110.

,;. . .Tyler, R. & Ayliffe, G. A. J. (1987). A surface test for virucidal activity of disinfectants:. r, prelimary studies with herpes virus. Journal of Hospital Infection 9, 22-29.

"

c'

-

Journal 0/ Clinical Patliology, 1978,31.923.928

A test for 'hygienic' hand disinfectionG. A. J. AYL1FFE, J. R. BABB, AND A, H. QUORAISHJ1From tli~ Hospital ¡n/ution R~s~arcli Laboratory, Dud/~y Road Hospital, Birmingliam, UK

SUMMARY A standardised test procedure is descrbed in which finger-tips are inoculated with brothcultures of organisms (Staphylocoçcus aureus, Staphyloccocus saprophyticus, Escherichia coli. andPseudomonas oeruginoso): counts are made from washings of bands after disinfection with variousantiseptic-detergents, alcoholic solutions, or unmedicated soap. 70% alcohol, with or without

e-hiorhexidine, was the most effective preparation. The two antiseptic detergents showed vaiableresults. but against Gram-negative bacili neither wa signifcantly more effective than plain soap.Some tests were also made on the death rate of organisms dried on the skin without disinfection.

Disinfection of the skin may be as either bymeuring the reducuon in numbers of natural(includi.ng reident) bacteria or of bacteria artifi.cially applied to the skin, that is, transients (pria:,19-38; Lowbur)' n nl.. 1964a; Männer et al., 1975).'Hygicnic hand disinfection' is now an ac:pted terin Europe for a mcthod in which transient organismsare kilcd; the tcrm 'surgical hand disinfecion' is

used to descibe methods by which organisms of thereidcnt flora arc removcd or kiled. Transientbactcria, including Stapliylococcus aur~us or Gram-ncgativc bacilli. are usually present on thc skin insmall numbers and can often be removed or redua:dto vcry small numbers by washing with sop andwatcr (Aylitfc et al.. 1975). Ncvertheless. in somecircumstance. a highcr degree of safety is reuired,and antiseptic preparations are need for thereliable killing of transicnt orpnisms.

.. Althou,h tests of cffcctivenes of disinfecants"'againsi artificially applied OTJnisms have often

ben reported (Miiicrmayer and Rottcr. 1975;Lowbury lt 0/., 196a). nonc ha ben ac:pted as asiandard test for antisepiic or hygienic hand disinfection in ihc Uniicd Kinidom. This papedesibe thc developmeni of a test modified from thoffcial Wesi German method (DGHM, 1972), inwhich the finicr tips are inoculated with a brothculture of the iesi organism. Thc efce ofvarious aniiseptic detcrgcnt and alcoholic prepara-tions on Stapli. aur~us, Stapliylococcus mprophy-

licu.t, Ps~udomo"as aeruginosa, and Escliericliia coliis asd in two cxperiments. and ihe devlopmetof thc lesl is desi bed.

Met and materls

APPLICATION OF TEST ORGANISMSBroth culture of various challcnic orgnisms wcreprpare by plating nutrient agar slope culturcs onblood agar. After incubation five colonies weretransferred to nutrieni broth (Oxoid NO.2) andincubated for 18 hours ai 37°C.

To remove superñcial transient bacteria volunteerswahed their hands wiih non-medicated soap andwaier (soial wash) and dricd Ihcm thoroughly on

paper iowels. Wiih the palms of the hand facin~upwards and finiers outstreichcd, 0.02 ml of thebrolh cuiiurc wa applied 10 the iip of each fingcr andthumb with a 'SO dropper' pasicur pipeiiC and wasspred by rubbing tOliher opposing fingcrs and

thumbs for 40 seonds. Th proc was complciedby dryiniihe finicn in the air. 1o'iihout rubbing; foranolhe 80 sends before sampling or disinfection.

Trst orionismsStaph. oureus (I) NCTC 9716, Staph. our~us (2)NCTC 8354. Sioph. mprophyticiu (Baird Parker M3Bio typ 2. novobiocin reistani). Esrli. coli (i)NCTC 8196, &ii. coli (2) Insi. Hygicne, Vienna,Ps. IIrUlinoMl NCTC 6749, Serratia morcrscrnsNCTC 2847. and an endemc strain of klcbsicllalCotyp 21 fro a urolOlical ward.

DISINFECTION AND WASHING OF HANDS

Recive for publication IS 101)' 1978

Application of ontisrptic tkterirnts and liquid soap

Two minutes afier the application of the orpnisms,the hands we moistene under a running iap, and5 ml of the prepation wa cafully poure into thecuppe hands. Th prpalion wa applied for 30sends by a standard procure. consisting of fivcstrokes backward and forwrds: palm to palm.

923

'Prni addre: Dcpanment of Medical MicbioloC),Uandouih Hospíiil, Cardiff

924

ri¡¡h! palm over lefi dorsum, lefi palm over rightdorsum, palm 10 palm wiih fingers interlaced, backsof fingers 10 opposing palm with fingers interlocked,rotational rubbing of right thumb claspe over leftpalm and left thumb clasped over right palm,rotational rubbing backwards and forwards withclasped fingers of right hand in palm of left hand andclasped fingers of lefi hand in palm of right hand:hands and \\Tists were rubbed until the end of the30-seond period, then rinsed under a running tapfor 15 seonds and dried, with two paper towels, (or15 seconds.

Applicatioii of alcoholic pr~paratio"sFive milliJitre of the preparation wa poure intothe cupped hands and rubbed onto th ild.e for 30seonds. The same procure wa us as with thedetergents. Another period of 30 lConds waallowed before sampling to allow the hads to dry.

RECOVERY AND CULTURE OF TESTORGANISMSAlter disinfection. the fingers and thumbs wereimmersed in 100 mJ of nutrient broth (Oxoid No.2)with added neutralisers contained in a sterilisedbowl (100 cm in diameter) and rubbed vigorously on50 ml of glass beads (3-5 mm diamter) for oneminute. Nc:utralisers added to the broth were 0.75 %lecithin-Twen mixture (50 g Twen 80 and 5 I oflecithin) and J % sodium thiosulphate. Aftersampling. the hands were rinsed, dried, and dis-infecied wiih 70~~ ethyl alcohol by the methodalready described. Ten mililitres of reovery brothwas removed and thoroughly mixed on a rotarymixer, and 0.5 mJ quaniities were transferr, by a

0.02 ml drOlipin¡ pipette, on to at leat two well driedculture plates. Surface viable drop counts were madefrom thes and appropriate io-fold dilutions of therecovery broth. All plates were incbated at 37°C for18 hours.

Th folJowinl culture meia we found suitablefor the reovery of test orpnisms: bloo ap(Oxoid Columbia ap ba eM331 an 7'5%hors bloo) ror Ps. Ml1iKiflOSD, Elh. coli, S.mQrcrs('~ns. and K/~bsi~lIa spp, nutrit apr (Oioid

No. 2 broth with i.2 % New Zcand ap) con-taining ) ~~ hors-sem and pheolphthalein di-soiumphosphate (Babe and Kupe, i 95 1) (orStaph. aur~us. and nutrient apr containini 4 ,.aJmI

novobiocn for Staph. saprophyticus.

SURVIVAL OF TEST ORGANISMS ON FINGERSBroth cultures of Staph. alirl!W, Esch. coli, Ps.aeruginoYJ, and Klebsitdla spp we applied to the

tips of fingers and thumbs as previously desbe.Fin¡e were sampled individually at two-minute

G. A. J. Ayliffl!, J. R. Bobb, and A. H. Quoraishi

iniervals for up to 10 minutes by rubblng on glassbeds in gallipots of broth for 30 sends. Duplicateswere obtained by samplin¡ one fin¡r from each handafter each time intervaL. .

A series of tests was also made with the sameorJanisms, and with Staph. SDprophyticus and S.

marClsctns, in which all the fingers were sampled attwo minutes in the manner previously desribe.

Comparisoii of antis~ptic preparations: 1Five preparations were compare by a latin squaredesign of experiment (Lwbury ~t al., 1960). Sixstaff volunteers (thre male and thre female) tookpan in the experiment.

PreparationsJ--nmicated liquid sop2-i % chlorhexidine detergent (Hibiscub)

3-7'5 % povidone-iodine sur¡ical scb (Betadine)

4-70 % ethyl alcohol~. 5 % chlorhidine iluconate (Hibitane) in 70. ¿ethyl alcohoL. The alcoholic prparations contained) ~ Jlyo:rol as an emollent.

Six experiments we made on Tuesdays andThu~ays for 3 weks. In every experiment all pre-pations were tested, and all subjects used eachpreparation once. Each subjec was also left un.treted in one of the six experiments. The applica-

tion of test orpnisms and disinfectants and the

reovery of orpnisms were as alredy described.Two series of experiments were made with the samepreparations and volunteers. but with differentorganisms: Staph. aureus and I's. o~ruginosu.

Volunteers were asked to wah their handsbetwen experiments with non-antiseptic prepara-tions. If disinfection was reuired beuse ofaccidental contamination (most of the volunteers

wee laboratory staff), '7% ethyl alcohol containin~i % Jlyo:rol wa used.

Comparisan of anti.l~ptil! preparations: 2Four preparations, unidicated liquid soap. chlor-hexidinc deterrent, povidone-iodine detergent. and70% ethyl alcohol, we comp against various.tet orpnisms in aroups of approximately 10volunteers. The test orpnisms were Esch. coli 2,Staph. o"r~us J and 2, aD Staph. saprophyticus.

Experiments with Staph. DI~lU J we diontinuedwhen two (one severe) of the voluntee developedsepsis due to the test strain, and a icond strain.Staph. SDprophyticus wa use ror subsuent tests.Whenever poible the same p-oup of volunteerswa used, but in this test method each person washis/her control for each preparation.Test or¡anisms we applie and reved in the

manner preiously debe, but a preisinfection

A

sa'pi'th.sat\lorarinarmirp

( c:8

n

grctri

f

c

)uoraishi

on glasIpJicaresch hand

ie sameand s.ipled a:be.

square.oj. Si~

:) took

idine)

n 70~ 0

- ~tained

's and./1 pre.

eachf¡ un-

iplica-d theribed.same

Teren!

iands,para-;c or

iieersjnin4

.hlor.and

rious, 10'¡i 2,'ieus.iued.)pedrain.eslS.:eerswas

thetion

A Irsilor 'hyi~ie' lt ¡Jitr/eelion

I,

i

I

¡Ii

saple wa taen from ea volunter on com-pleion or inoclation and drng. The hand wethen rinse under running water to reove thesapling broth. contaning neutraJisrs, dried ontwo paper towels, and then reinoculated with the testorpnisrn. On completion or the seond inoculation

and dring peod the hads wen: wued or diinfeced in the standad maer alredy desbe,and the survivig orgisms were revere. Thme logaithc reuction of each test orgismin the group of volunteers wa used to compa theprpaations.

.~~":':

925

iignifcatly mon: efective than the antiseptic deter.,ets. The antiseptic detergents were not significant-ly better th unmdiClted soap in killing or remov-ing Ps. øeruginoso.

An anysis of variance (Table S) showed signifi.cat differenc between tretments but not between

c:perimcnt& days in both series of experiments. Thedifference betwen persns was significant for Siaph.oureu. but not for Ps. øeruginoso.

Table 6 shows tht consistent results were obtainedfor unicated soap and for 70 % alcohol in the

seond study. Similar reults wen: obtained in repettests on leveral or the preparations. 70 ~~ alcohol

wa iignifcantly more effective than unmedicatcdioap. but other relJlts were variable. Chlorhexidine

dctergent was significantly more effeclJve than un.meicated sop or povidone-iodine against Sioph.MlrOphylicu.. Povidone-iodine was significantly moreefective th unmedicated sop and chlorhexidinedcter¡ent &pinst Sioph. øureus. There was no signi-fit differnc betwen the antibacterial detergent

prpations and unmedicated soap when E. coli iwa the test organism. Isopropyl alcohol showed asimilar reduction to 70 % ethyl alcohol with Siaph.

mprophyiiciis as the tcst organism.

DisnIn the development or a standard test method,several factors must be considered, for example, siteand method of application or the organisms,

Table i Survivol of tesi or,onisms on i~ skin of ilit ¡;nrer.iips

T~s, orlllIlS'" 1,,¡iiiI S-.i... 1"'-") II,P.--_'''h. ~IU (I)

(..-1) i .,. 4_ 6 ,.¡" , """ 10 "'":

JI ,,)6 6"2 ..13 ',50 6-( 6').we; 1.23 '.11 ,~ N' 6.1- 600

úc. col, JB '.11 ,~ ,-0 .,. . )0 ) 01we; 72, 6.'. 5.9) ..1) .75 .2'

1". en",'~ JI 1.15 ,. '04) ."7 ..'0 ..72we; 1.)0 '7-0 H5' '.2, ,.2 ...,

~"'.I" "". JB .,2 ,.0 '04' 5-, "01 '.11wO "S2 Hi 6. 5-1 5-5 5-,

..~

.Ia all Cl ._ *Y i- _ li,, tb i_tn_ ~

T,..iw N_ /a,;. ... _,Table 2 SIIifl of lUI M'tmS Oft 1M ¡;",ers ofter t"" iitu

No. .I_".'..".u No. .firt, lJIIH

,II..52II)1"

.

.

.,,.101519

N_........",.J_

e .t5ItS .A large proporton or £Sch. coli and Ps. Q('rii!inosoapplied to the fige died during a period of i 0

minutes (Table l). Klebsiella showed a muchinter surival. and the numbers of Sioph. olirellremained alos uDCn,ed after the initial peiodof drying of two minutes (Ricketts ('i 01., 195 I). Allthe cultufC appered to be viibly dr withn twominutes, although this varied between 30 seonds andtwO minutes in different individuals. Table 2 con-firm' that an approximately iQ-fold reuction in

these three organisms, S. morcrscens, and Siaph.

soprophylieiis ocurs within two minutes.The mea log counts arter the various treatments.

in the firS1 compaative stud)', are shown in Tables 3and 4. An treatments were signifcantly more cffectivethan no trtmt and both alcoholic iolutions we

S. ",C".rrru". anc,ll&rh. coli i&di coi iS"". ~"l& JS,aplt. -.rll i

~'oPlI .lr-~I,C~

....

....,..1.27..1-..""16

i.eii0-12D.'71'5)0-1-0-150-79

)uoraishl

on ¡lassiplicatesch hand

ie sameand S.ipled atbed.

square01. Siir=) took

-¡dine)

n 70 ~~

-rained

'5 andi11 pre.

eachf: un.

,plica.d theribe.same

ferer

und.pa.a..e of

!leers.inin4

tilor.and

rious10

1¡ 2.'1eus.

iued)pedrain.ests..eers""as

thetion

A I~st IDI 'linlinie' Jt diinfution

e

aaple wa Wen (rom eah voluntee on com.pleion of inoclation and dring. The hands wethe rins under running water to reove theI&pJin¡ broth, containing neutra1isrs, dried onrw pape towels, and then reinoculated with the testorpnisms. On completion of the seond inoculationan dring peod the hads were wased or dis-infeced in th standad maner alredy desibed,and th survivig orpms were reve. Thme logithc reuction of each test orgaismin the group of voluntee was used to compa theprations.

I

I.esltsA lage proponion of £Sch. coli and Ps. ,"ruginosaapplied to the fi¡e died during a peiod of JOnùutes (Table 1). Klebsiella showed a much

irter surviva, and the numbers o( Staph. aureiisn:ined alost uDChan¡e after the initial periodof drying of two minutes (Ricketts ~I 01., J 95 i). Allthe cultures appered to be viibly dr withn twominutes. although this vari between 30 seonds andtwo minutes in different individuals. Table 2 con.fi thai an approximately IQ-fold reduction inthes three organisms. s. mQrusc~ns. and Staph.

soproph)7iC/ls òcurs within two minutes.The mean log counts after the various treatments,

in the firs compaative study, arc shown in Tables 3and 4. All treatments were signifcantl)' more effectiveth no trtmnt and both alcoholic solutions were

. "

925

Ii¡nifcatly more efective than the antiseptic deter.aets. The antiseptic detergents were noi significani.Iy better thn unmdicated soap in killing or remov.ina Ps. oeruginoso.

Ai anysis of variance (Table S) showed signifi.

cat differnce between tretments but not between

expemental days in both series of experiments, Thediffernce between persons was significant for Staph._ell but Dot (or Ps. Q~rUKinosa,

Table 6 shows that consistent reults were obiaine.dfor unic:ted soap and (or 70 % alcohol in thesed study. Similar reults were obiained in repeitests on several of the preparations. 70 ~/~ alcohol

wa sigiuc:ntly .more effective than unmedicatcd

sop, but other reults were variable. Chlorhexidine

detergent wa significantly more effective than un.micated sop or povidone-iodine against Siaph,Itropliylici. Povidone-iodine was significantly more:efective th unmecated soap and chlorhexidinedctericnt &&inst Srøph. aiireus. There was no signi.fit difer betwcn the antibacterial detergentprepations and unmedicated soap when £. coli 2wa the test organism. Isopropyl alcohol showed asimila reuction to 70% ethyl alcohol with SlUph.

IDpropliyliclis as the test organism,

DisOlIn the development of a standard test method.sevel (actors must be considered, for example. siteand method of application of the organisms.

Table i Sur"il'1 of iesi of7ønisms Oil tlie slen of ili~ finie'-tips

T.lt .,."um 1'';,., s...._, (., _II) ",n.-w_'...-.IU(I)

(io-') 2 Mi"- 4_ , ~ "'ÙI , ..'ii 10 If,if

JB '.)6 6'92 "13 6-50 6.66 6.)0we ..23 HI 100 H' ,... 6-o

kl. to. JB '.11 ,-o ,-0 ..., ..30 HOwe '2' 6". ,.,) ..13 ..,) .2',..._- JI ..1' 1il Joi .." .'50 ..72we; .30 1-0 '-61 "2' ,.2 ...)~i"lP. JB '.'2 500 ,.., '.1' HII ).11we "'2 H) ,-0 '..1 5-, 5-)

... al _ .... .... _ Iior \i ,_""nutf ..p1í..

Tabl 2 SI¡",¡ of iut orøtims Dt 1M fi",ers liier lwo MuuTui__ N_ útlllo -, Nr- lo. ..,.. .tNP 1 .. No. .1 __..,it Iro. "1"" ",Stilt

S. -..l'' ... i.eii , .i'. ....'1I ... 0-.2 II 1~ftlil ,.. 011 t. ,ud. røi 1 '.2' 1.'3 '2 I.~.__rlUt .... 0-.. II ID

~,_rtQ1 ..)) 0-1) ii I'S~. _røy';cou 1.16 0-"7 6) "

Table 3 Vjabl~ COllflts (lors) of Staph. aureus f,om fillu.iips

926 G. A. J. Aylifft. J. R. Babb, and A H. Qunraishl

£c~, 1"1',.1 lo ('"' Lø l"'" .f'~ ,,.,..,.,., ..¡,it.(11 IrH'..,.') s- C./orll,ziduw ".....lI,rw 70~. ,iii"' alcol (J $~. Ull O"Ø/I(

.,"'.,"' ..,.,..., rllorltrzuilftt'

7-6 (TBI HOOB) 6.11 (AO) ~.2) (LTI '.50 (LO) ,..ø (COl

7.90 (COl 7,. (LTI ~.,i (TBi ~.2) (AOi ,-0 OBI ,.(, (LGI

7.12 (LO) 6-0 ("0) ~'60 rCD) )~ (TB. ).90 ILT) ).60 UBi

,.6) UB) ~~(TB) ~.2I (LG) 5-16 (COl )010("0) '.'OILTI

,.7 (LT) H) (COL ~2S OB) '4 (LG) '.21 (TBI ) 581"Qi

1.2) (AO) 6-0 (La) '4 (LT) '.1' UB) '4 (CD) 2.90 (TBI

'.1' 6.11 ,." ,+I ,,0) )-7

i2),~6M.." lo cou'U

llUuals or &ubji an .i"Cn in llftlha

Table 4 Viøb/t coiints (lors) 0/ Pi. aeniJinos from fitltr-ips ( C£,i. '.¡,iGl .. t'.., i. eetJ .'"' ,~'" ."It.

(""__"'1 S- CIH#WZ..1W ,.--iM 7D~ "II" .¡to o. $ X -',øho/ii".."' ","I t'IMltrxuJ,.

1 "19 ("i ,." (11) 4.1) (OA) '.'U (Ll) ,"0 (l0) '"') (CO)

2 '~(CDi 4-6 (L Tl 4')6 (") _(OA) 2.'1 (JlI) 4.26 (LG)

) ,.1 (LOI 4-0 (GA) ''' (COl ,.)0 (T) 2-0 (lTl HOOB)

4 6.1) OBI 4,11 (T) 4.. (l01 4''7 (CD) )-0 (OAI HO(LTl

~ 1.11 (LTl 5-1 (CDI 4-9 UB) H' (La) 4-6 (T) ).)1 (GA)

6 '" (OAI ,.)1 (LOI 4.12 (LTl "21 UB) 2-9 (CD) HO(TBI

Maii ..COIII '4 ,.2. 4," 4.71 ).0 3-6

i..itiail or aubje arc ,¡9C ¡.. paftl_.

Table S Åflalysis (If ~Qrit,.n of /01 Ctts o/Staph.au1"US a/,tr t'ta,mtnt

Dq'OfJ 011_ MHI_"-.. ~bpcnmcnu ,T..unenu 5Jl..dual 20

0-54" S (p C O-~ ;, 0-2)D-J0 NS

13.1751 S (' "' 001)0-2010

.A_lysis oj .arulfrt 01/01 COllIS 01 Pi. acJinos tI/rrr

t'eDt_nt1)rwJ .11.. 11-_Peii ,E._.." ,T_lftu ,

....1 20

..,'" NS

...20 NS.4-014 5 (. C 001)

..2 I CI

mehod of appliation of the disinfecnts. andmehod of revery of the orpnisms after dis-infecion.

The choice of a tes orpnism dends on itssurvival on the skin, safety, ca of identifcation onrevcry meium, and reponse to differnt dis-infects. It wa conside that at let one Gram-~tivc bacillus and one Gram-poitivc coswold be reuired in a tesl.

Most Gram-negativc bacili die rapidly on drying.but £. coli showe a consistent number of survvors

after drying on the skin for two minutes and iswidely use in test methods in other countries. i i was,therefore, included as the main test organism.

KJrhs;~lIa atrogrnrs survivcs better on the skin than

most Gram-negative bacilli and. in view of iisinc:sin¡ clinical significance (Casewell andPhillips. 1977). might be considered as a possiblealterntive test orpnism.

Staph. tllirrU$ is more sensitive to sóme anti.setÎC. for exmple, hexachlorophane and chI

or.hexidine. tha Gram-neptive bacill and wasinitially chos as the othe test organism. Itsurvive well on the skin but could not alwavs be

eaily distín¡uishc from othe orpnisms i~ theredent flora. Th phosphata test wa satisfactorybut reuir some experince. Prliminary tests wit h

a tetraline-reistant strain show slight in-hibition whe the reve meium contained 10~&lml tetrdine. Howe, Siøph. liurrliS was con.sided unatablc wh two subje developedaeis of the ftnsers in the seond sees of tests. Anovobiocn-restant. weJl-pi¡nted strain of Staph.Stprøpliylicus wa chos as a safe alterntive toSIØPIi. tI,trrus. Unfonunately, it wa more sensitivethan Staph. aurriis to chlorhexidine detergent in bothin-vivo and in-vitr tests and wa therefore not

suitable as a test orpnis. Further studie on other

f

orQishi

~;cW/._

));¡,

)),.J

A tesl for 'hyK~nic' Iid diinection

Table 6 M~all /oraritlimic re4CliJs of iesi or,atWs a/ier Ir~aim~Il'

927

Mnu Ie "",;0 f~iøn 111" '..'-.1 (No. el "¡,.ciil

So Clli.... .'rf.'" "'lI""'" .,""", 70 X "~J" eJr01

S,.,._I )'67 :: o.n(10)

SMR. '-iu i 2.)1 :: IHI 2-4 :: ø- ).02 :t 0-.1

(10) (Ii) (101S...~,;nu 2..9 :: 0-.) ).71 :t 0-69 1"9 :t 0.)1 )-'2 :t 1-07

(12) (10) (10) (II )).71 :t 0-62 2'" 2: 0..' 0).' I :: 0.12

(Ii) (II) (10)

EM.. coi i 2..1 :: 0-., 2.11 :t 0"6 2.76 2: 0.'1 )-4 :: 0.79

(10) ( I) (10) (10)2.16 :: 0.7)

(10)

e e la.propyl alcohol. Siaftdard *,...iion.-'-,r- -.,.

-,

d is-"'as,

IS....han

itsand,ib~

nti.lo~"'as

Itbe

(~ory-jihin-10

)n-Je

AI1h.

10iveIthlOtier

orpisms to fid a suitable: alte:rnative to Staph.Iil1rew ar nery.

The: method and time of application of the dis-infeclant are also importnt. particularly withalcoholic solutions. In our studies with 70X alcoholin the: clinical situation some uneplained highcounts were obtained after tretment. Similar high

counts-w sometimes obtained in our preliminarylaboratory experiments. Tests made with an alcoholicdye solution indicated that are of the hands oftenremained untreted by the: solution, paicularly thetip of the thumb (Taylor, 1978). A standard methodof application is. therefore, reuired for testing. butfor routine purpo a les complicated method than

that dcsibed for this tet would be nesary.Although the: studies with a dye suggeted thatadequate cover of all the sunace of the hands wasnot nerily related to duration of application,

the duration of application of the disinfectant also

ha some relevance. The men tim for nurs',aandwashing in wards was found to be 21 icnds~nd ranged from a few seonds to )08 seonds.

Thiry seonds seme to be a renable com-promise. and our unpublishe studies andthos ofothe workers (Lowbury eiiil.. )96b; Miueryerand Rotter, 1975) indicate that efecivene is notsrt1y improve ir the time is inc to one totwo minutes.

Saplini of the finae-tips is eaier thn that ofmot other sites. and consstent reulu have benoblaine by othe workers with this method (Rotter

rt ill., )974). Finier-tips are als freuently con.taminated in clinical praice. Th numbe ofsurvvini bacteria paiclarly arter alcoholic dis-infecion. wa often very smlJ. Samplini the wholevolume of wahinJS with a membrane filter ispoible, but when this method is us a hevyIJowth of the norml reident flora is obtained.Rubtiing the fingers on the base of a petri dish in 10

ml of broth al improve the sensitivity of the tesi(Rotter et ill., )974), although unpublished observa.tions showed that the bowl and petri dish testusually pve comparable: reuhs (W. KolJer and J.R.Babb. persnal communication). Agar contactplates. usd instead of wahings for reovery oforianisms, ar usful for a quick a.sment ofefecivene and for in-use clinical tests (Smylie ri 01.,1973; Ayliffe et ill., 1975) but are insuffciently

stadardise for a comparison of preparations.

Th menini of any laboratory iest in clinicalter is diffcult to &Ssess. Organisms applied to the

skin artificialJy may behave differently from naturallyacuire orpnisms (Ayliffe and Lowbury, 1969),and the method of application may affect theefciecy of disinfection (Lily and Lowbury, 1978).

In ward studies. hand sampling after the use of soapand water or 70X alcohol gave results rathe:rsimilar to thos obtained in this study (Ayliffe n 01.,)975). Pathogens are not often prent on the fingersin la numbers, but lot to 10' Siaph. aiireiis andGram-netive bacili have bcn reovered fromnurs' hads after variou~ nursing procdures. Areucion of 101 (99 X) obtained with sop and water

(Loury eiiil., 196a) is probably suffcient for mostpurp, and Gram-neative bacili die rapidly ondry skin. Th antisptic detergents were more effect-

ive thn sop apinst some organisms and may have

lOme value in speial units, for example. speial carebaby uniu. 'OX alcohol with or without chlor.hcidine is more efective than the antiseptic deter.iinu but reuire care to ensure loo coveraJC ofth hands durini the period before evaporation.

A Ildard te that meure the men 101reucion in numbers of organisms can be inter.pred in differt ways. dependini on t~ beliefs ofthe country or pen concerned. However. anintertionally ag interpretation should be

poible: when suftkieni daia are available: from

928

sampling hands of staff in clinical ar. A com.parison of a standard sop and water wash and anapplication of 70 'X alcohol with that of an unknownagent should provide useful informtion. A test(Rotter ~I 01., 1974) similar to the one desibed herehas ben propo!id for poible us in Europencountries; it is hoped that other laboratories wilcarr out reproducibility studies on one of thes tests

after an)' necessary modification.

We thank Dr' M. Rotter, Institute of Hygiene,Vienna, and Dr R. R. Marples, Public Health

Laboratory Service, Colindale, for providing uswith two of the tesi strains (E. coli and Staph.saproph)'iciis); the staff of the Hospital InfecionResrch Laboratory and the Microbiology Depart-ment, Dudley Road Hospital for taking part in thestudy; M. D. Wilkins. FIMLS, MRC IndustrialInjuries and Bums Unit, Birngham AccidentHospital for help with the statistical analysis; andDr E. J. L. Lowbury for advice.

ReferAyliffe, G. A. J., Babb. J. R., Brid,e, K., Ully, H. A.,

Lowbury, E. J. L., Varny, J., and Wilkins, M. D.(197.5). Comparison of IwO mehods for assini thereoval of total orpnisms and paihoaens from theskin. Journal of Hytirnr, 75, 259.274.

Ayliffe, G. A. J., and Lowbury, E. J. L. (1969). Soura ofps pnirene in hospitaL. British Medical JourlUl, 1,333.33;.

Barber, M., and Kuper, S. W. A. (19.51). Identification ofSiaphylo('o('cus pyogrn~s by phosphatase rection.Journal of Pathology and Bacirrioloiy, 63, 6.5.68.

Ca_II, M., and PhilipS, J. (1977). Hands as route ofiransmission for XlrbsirIJa speies. British Mrdi('alJournal,1.131.5.1317.

Deuische Gesellschaft rLir Hyiiene und Mikrobioloiie(DGHM) (19721. Richtlinien rur die Prüfun¡ chemi.scher Desinfekiionsmiiiel 3. Aufl. G. Fiscer, Stull.

¡ari.Lill)', H. A.. and Lowbury, E. J. L. (1978). Transient

skin flora. Their reoval by clcnsni or disinfection in

G. A. J. Ayliffr, J. R. &bb. and A H Quoruishi

relation to thr mode of depoiiion, Journal of C/"".

eil Paihology, 31, 919.922.

Lowbury, E. J. L., Lilly, H. A., and Bull. J P (19NllDisinfection of the skin of opçrai,on siies 8mo1lMrdiail Jourftl, 1, J03~.

LowbuTY, E. J. L, Lilly, H. A., and Bull. J P. (1964a iDisinfection of hand~: Removal of .ransieni organismsBritish Mrdical Jourftl, 1, 230-233.

Lowbul'., E_ J. L., Lilly, H. A., and Bull. J. P. (1964blMeihods for disinfection of hands and operation siiesBritish Mrdical Journal, 1, .531.S36.

Männer, F., Roiier, M.. and Miiiermayrer, H. (I97S¡.Vcrlcichende Unteuchungen iur Keimgewinnung vonder künsilich koniaminiener Hand. Z~nlralblo" JIIIJktrriologir, ParasiirnJund~, Jnf~k t.onslcank h~,,~n.

11M Hy,irnr, Abt. I: Orii. 8160, 412-43 i.Miiicryer, H.. and Roiier, M. (197S). Ver¡ieich de(

Wirkuni von Wasser, einiien Dcterieniien undAthylalkohol auf die uansienie Flora der HandeZ",lra/búJf fir 8aktrrialog;~ Parasii~nk und~, Infrl..

iiOfkrllliriten ii,, Hnirnr, Abl. I: Orig. 8160,

J63-172.Pr, P. B. (1938). Tl bacterioloJ) of normal skin. a

ne quanlitaliYe test applied 10 a study of the bacterialflor and th disinfection action of mehanical clcans-

íÌl. JDlNlI of J1trclious Disras~s. 63, 301.318.Ilkett., C. R., Squire, J. R., Topic)., E., and Lill) , H. A.

(19.51). Human skin lipids with pariicular reference toth self stmlisini power of the skin Clinical SClrnct,

JO. 89-111.Rouer, M., Miuerye, H., and Kundi, M (19741

Unicruchunaen zum Modell der künstlich koniam,.nierten Hand: Vorshlai rLir cine PrüfmethodcZrntrDlblaf/ jìir IJktrriolo,i~ Parasiirnkund~, /nfd.iiOlskrDnkhritrn und Hnirn~, Abl. I.' Driii. 8159, 560S81.

Smylie, H. G., Logic. J. R. C.. and Smith, G. (197)1From Phisohex 10 Hibiscrub. Briiish Mrdi('al Journal.4, .58~.589.

Taylor, L. J. (1978). An evaluaiion of handwashin¡technique. Niminz Tiwws, 7~, S4.SS, 10S.110 e

Requets for reprints to: Dr G. A. J. Ayliffe, Re¡¡,onal

Health Service Infecion Resrch La bora i 01' . Dudle~Road Hospiial, Dudley Road, Birminiham BIS 7QH

~I Designation: E 1115 - 91

..;:"l.,:.:..

----.. - - Standard Test -Method for--'':::_-.:~:'Evaluãtion'of Surgical Han~ Scrub Formulatipns 1

.-. _...'. .... .. -..... ....... ._---- . ------. _. . .- - . -- -- .. --_._-_.- -.. s;ò\:

. . _..' -_. "- -'.-.' - _._..._ 9"'~¡ .'--~:;_...Th si is is uDder the fi ~~~ E IllS; the Dumbe imedy follow th deoD iadica the )a of

.0ri adOD or. ia the ca of reoa. the )a of la reon. A Dumbe ia patl ia the )a of la reva A .Slpm epoD (i) iadica an edri di sice the la reoD or reva

,.iIl

!

1. Scpe-.1. i' Th te metod is degned to evauätë å.ticrl:úiagents in formultions for utity and effecvenes as surcahad scbs It is intende for detg both i,edtemicrbia reuctons and reuctons with re us (re.sidua effec).~r.; _ .

,-c NOT . I-A Iaowed of micrbiologca teue is reui fortI prum~- ... .'1::1.2 This stad does not purport to adress all of thesafet problem. if any. associated with its use. It is theresponsibility of the user of this stand to establish appro-prie safet an health praaices an deerine the applica-

büity of regory limitaions prior to use.

,

t'!I..;

2. Re(ereoce Doeotsi.l ASM Stand: .E 1054 Prce for Evaluatig Invars of Anticr. bi Agents Use in Diect, Sati, Antitic,or Pred Pruet

2. Othe Doc:Stada Metod for the Extion of Da PructJ~~M ~~ç M~ac ~19654 .~;~Så o(T~i Method . ,::' 3. I Th te metod is conduct on paèi Selecfrm a grup of volunte who have reed frm usgany anticrobi for at lea two weeks pror to intition ofthe te At lea twelve paeli ar selec frm th grupon the bas of high inti ba count, 1 x l()5 pe hadas deed by balie meaents of the ba onthei hads .. . ~.' . ;~'3.2 The selec pa peor a siul suca

.. sCb unde the supeon of an individua compet inasc tehnque. One-th of the paeli' hads arsapled immedy af the scb (wi 5 mi), one-. th af 3 h and the reg hads 6 h af scbbig.

. No mor th one had of a paeli is sapled at a giventie inter.' '. .:-.3.3 Ten adtiona scbs ar peormed with the te

i Th le metod is unde the jurctoD of AS Commi E.35 ODhsci and is th di rensbity of Submmitt E35.15 OD ADticrbi A¡tsCut edOD apve July IS. 199 i. Puli Sept 199 i. Ory

puli as E IllS - 86. La prous edtin E III S _ 86.:. i A1 Bok of ASM Slds. Vol

I 1.04.3 Avae fr Amer Publi He Asoi1D Wasa. DC.Qi Stda Pl Count Metod4 .uTCC Tes MÐi 1968 Tec Maua SeOD 8-175. ava fr

th Ami: Asoa of Texe Cl and Colo P.O. Box 12215.Re Tri Pa NC 27709.

"18

i .;u.è

formultion over a 5-dy peod followig the inti sc.

The hads ar sapled two adtiona ties; once af thsend scheduled us of the prouct and ag af the lascheduled scb. ._-- ... . ~ ....

4. Sigc:c: ao Use

.. .' ..:S~4.1 The procur in th te metod should be Us toevaua the abty of a te formultion to reuc theba popution of the hads imedtely af a .sîeand multiple us and to dee the trd in gr ove å6-h peod af sie æ.d m~tipl~ us. . '" ..~~ .;!-

::',::.~5. Appatu , :.;.~fJ~5.1 Colony Couer-Any of se ty may be us

for exaple, Que Colony Counte. ..... .~:~jf5.2 Incator-Any incubator capale of mata ä

tepetu of 30 :! i-c may be us " .5.3 Sterlizer-Any sutale st st cale of

prucg the conditions of stty is accle. .';. "'i,;:5.4 Timer (stop-lock), th ca be re for miut aísends. . ':. .. .' ~.... . ¡ .Gi _.5.5 Han Washing Sink-A si of sucient si;tó

pet paeli to wa without touchig hads to ,p.su or other .."el .... . .,. . ';. ;~/i":.r- . _".. . '_~ ,.5.5.1 Water Fauet(s), to be loc abve the si aïaheit tht pets the hads to be held higher th Ui

el~ws durg the w3g ~ur. (It is delt fgr,~height of the fauces) to be adJusle.)" ,.' -.::;J:""'". 5.6 Tap Water Temperature Reor an..TemperatrMonitor, to monir and re wa tepe to 40 :ii-c. -'" ...':, '.' .. .., '. ~t;~

. ,;: . ;." ':'::g~~6. Materi and Reen _ . . ",. .. ~C .llQ... ~. 6.1 Petri Dishes-ioa by 15 mm. Reui' for ií.

formg stda plate count. 5 'i:';:':':':JQ2 ..6.2 Boaerologica Pipet, 10.0 and 2. or l..mLcapaty.6 . ,., '.' . ... . .=-~2n=~. 6.3 Water-Dilution Botdes-Any stle gJ 'èöii=taer havi a i SO to 200mL caty and tit clOsmay be us 7 _' '''',

6.4 Baseline Control Soap-A liquid cae soap or otherliquid soap contag no anticrobia. . .. .. . ......;

6.5 Gloves-Sterie loo fittg gloves of latex, un~

800

. -.'.., Prdíle pIc pe di ar aYa fr II JoIa suly Ii .'. . . '.6 Prdíle lioø pí ar aYa fr mos toia suy hous . .7 DiutOD boes of 16(mL c: bavi a sc-c cios ar ava

fr Coni Gla Co.. Kibl Gla Co. or mos tolarary suppy bous

259

pong no anticrobia propees.86.6 Test Formulion-Dions for us of te formul-

tion should be included if avable. If none ar avale. usdions prvide.in th te metod (se Seon 1 I). .6.7 Sampling So/ution9-Dilved 0.4 g KH2P04, 10.1 g

Na2HP4 and 1.0 g islphenoxyplyetoxyetoPo in1 L died wate. Adjus to pH 7.8. Dins in 75 mLvolumes into water diution howes or other sutable con-taer and st for 20 mi at 121-C. Include ananticrbi invator spc for the te formultionbeg evauate in the saplig solution us to collec theba saples frm the had followig the fi wawith the te formulon. , '

6.8 IMPORTAN-A defitive 'remmendaon re-gag the incluson of an invar in saplig solution(6.7) us for ba collecons pror to the fi wa canot be ma. The followig two points should be consderin mag a deon: (1) If an invator is included in thesaplig solution us pror to the fi wa wi reduainvator on the sk reuc the efca of the teformultion in subsuet waes and re in higher thexpe ba counts? (2) Ca saples collec withoutan invator be pr quickly enoug to avoid de-cr ba count due to contiued acon of the teformultion? Wher the deon, to fata the compa-isn of rets ac stdies the investor should indicatewheter or not an invar ha be include

6.9 Dilution Flui-Buttel'sll phoshate buferwate adjus to pH 7.2 and contag an anticrbiinvator spc for the te forultion. '6.10 Soybeon-ciein Diges Agar2, with supplementa

polyste 80 (0.5 to 10 gI) to stul grwt oflipophic ornisms. ..6.1 1 Fingerl Cleaing Stick, such as Pr-o Pr-

mium Nai Ceaer.13 .6.12 Sterle Han Scr Bruhes14 (reui o~y if sp-

wed for us with te formultion).

7. Tes Paeli7.1 Pael sh cons of hethy adult volunte who

have no clca evdence of deto have not reveantibiotics or ta ora contrve two we pror tothe te and who ag to ab fr thes ma untithe concluson of the te

8. Prpation o( Volaote8.1 At lea two weks pror to st of the te enll

· A si g1cn I' 8873C (si) FI 1. Pr G1CMfr Amca PI Ub GI CA 9120. A zo ofiitiOD !C su as AATC Tes Met 901965 may be us to evaitiaobi pres of glove .

9 Pen. A. F.. '"e Miaoolog of th Ha Evaua th Efec ofSiica Scbs. Deelopm in In. Micbiolog. Vol 14, pp. 125130.

'0 Triton X.lOO. avale fr Rohm an Ha Co.. Phphi PA.ii Biidd's Phosli Bufer. Jou of lM.. of Ofic Ag_luraJ Chm. Vol 27. 1939, p. 625.12 Uni Si P/i XX Uiu Sta I' Conv=tion.

Inc. Ro MD. CJ Mi Li TesiJ Pr Pr Nal Oc (P), Pn No. 8014-12. Muuî

ti by Davi aD Ge Ub, OD CM St. Da, cr 06813.I. A sule br Ha Sc Bni Woo No. 3390. is avale frGra F'el Surca Co.. Inc. New Hyd Pa NY 1104.

4t E 111'5

appxíteJy 20 volunte as potenti tes subjec. '8.2 Inct the voluntee to avoid conta with anti-

crbia (other th the te-formultion) for the durtion of

the te Th recton includes anticrobi-contagantiJ)ÌIts deorats shpo lotions sops andmate such as acds ba and solvents Bathg inchorite pols ånd hot tubs is to be avoided Voluntear to be prvided with a kit of nonaticrbi penaca pruct for èxclusve us durg the te and rubbe

gloves to be worn when conta wi anticrobia ca notbe avoide ..: :;..; ..':~ '.-: .. . .' '.;. ..-'.;' . .' '.. ",~,":(; ~ . '(::.~:: ". ,..i

9. Prur _.~. .", . . . : ; i : . ~!Ù Af piéiï have refred frm usg anticr

bi fot at lea two wee peor wa with baecontrl so (se 5.4, and Seon 10). Volunte ar not tohave waed thei lWds on th day 2 h pror. to baedetion., . Af wag, dee fi es ofbae ba populon by saplig hads and enu-mer th ba in the saplig solution. Th is Day iof "Baseline Perod" Re th balie detionpr on I;ys 3 and 7, Days 3 and 5. or Days 5 and 7 of"Balie Peod" to. ob th es of baepoulon. Af obt the fi and send es ofthe bae populons seec as paeli at lea twvevolunte who exhibite at, eah saplig inte counts1 x 10'. The th es of the bae popuon,obted for ea of the twve seec subjec ar ave-

ag to obt the mea bae counts '9.2 A bac radom sapli pla should be follow

The numbe of pael and saplig ties ded on thete forulon bu mus esli the on and ext ofthe ba su~on an the durtion of suonbeow the balie counts Equa numbe of paeli shouldbe ased for sapl tie. dr and hadees Atyca bace radomion pla for teg a block ofpael follows, ,. ... . 'PI No. Po 8a S8 Tir. tiOo .. 3-, .' 601 11 Iw,. ' 2'.' :,'ii Iw . _. .3 ri iw _. 11 iw, .. , rt iw - 11 Iw

.. . 5. . .'.. ..... . 11 iw . . rt iw6 .. rt iw : _ ..;.: 11 iw. 9ii The numbÍ..of paeli pe block may va bu

mus be dele by two and. by the numbe of saplig. ties in ord to asgn equa numbe oflef\ and right hads

to ea saplig tie.. :.. . ',' .9.2.2 Th mium 'numbe. of pael ded on

vaty encounte in the stdy and relave efca ofdr Use ofles th twelve paeli each pe dr is notadvi for fi pruct evauations. In usng laer num-be of paeli it is only nec to incr the numbeof bace blocks: , :',: i .9.3 No soner th '12h.nor longer th 4 days af

completon of thei bae deteon, pael pe-form inti scb with the te formultion. Dee,acrdg to the raom saplig pla ba poul-tions on the paeli' hads at the asgned sapliginte (0 h. 3 h. 6 h) af scbbing. Deere baeri

260

rt iw.' . _ it tw

801

popultion by saplig hads and enumerating the bacteriin the saplig solution as sped in Seons 13 and 14.Repet th scbbing and saplig prour the next day

(Dy 2). On Day 5, ret the saplig prour aferscbbig with the te materi two adtiona ties on Day2 and th ties per day on Day 3 and Day 4 with at lea a

1 -h inter between scbs. Perorm one scb on Day 5prior to saplig. In sum, the paeli scb a tota ofeleven ties with the te formultion, once on Day 1 and

Day 5 and th ties pe day on Days 2, 3, and 4. Collec

ba saples followig th of the eleven scbs. Collecthe saples followig the sigle scbs on Days 1 and 5 and

followig the fi scb on Day 2. Th mics tyca useand pets detertion of both imedte and longer-te reuctons

10. Washi Tecque for Balie DetermtioDS

. 10.1 Volunte clea unde figer with na stckand cüp fige to s2-mm fr ede. Remove al jewelfrm hads and ar10.2 Ri hads includig two th of fore unde

rug tap wate 38 to 42°C for 30 s. Mata hads hierth elbows dur th prour and st outled in 10.3,10.4, and 10.5.10.3 Was hads and fore with balie contrl so

for 30 s usg wate as reui to develop lather.10.4 Ri hads and fore thorougy reovi al

lather, for 30 s unde ta water.10.5 Don rubbe gloves (6.5) us in saplig hads and

;e gloves at wr

11. Suc: Scb Tecae to Be Use Pror to Bacteric'. Sapli: '11.1 Rept 10.1 and 10.2.

'. 1 1.2 Perorm suca scb with te formultion inacrdce with dions fued with formultion.

NOT 2-If DO inctons ar prvide wi the te forultion,us the IO-mi sa pr in 11.3.

1 1.3 Ten-Minute Scr Predure:1 1..1 Di formultion into hads1 1.3.2 Se and st tier for 5 mi (tie reui for the

st in 11.3.3 thug 11.3.7).1 1.3.3 With hads dibute forultion ove hads and

lowe two th of fore..1 1.3.4 If scb br is to be us pick up with fige ti

and pa under tap tQ we without rig formultion frmhads.

11.3.5 Altively scb rit had and lower two thof fore and left had and lower two th of fore'. 11.3.6 Ri both hads the lower two th of foreand the bru for 30 s.:1 1.3.7 Place bru in strie di with ea reh.

'- 1 1.3.8 Repet 11..1 thoug 11..6 so tht each hadand fore is waed twce. The send wa and rishould be ümte to the lower one th of the fore andbe hads.1 1..9 Perorm fi ri. Ri éah had and fore

setely for 1 mi per had.11..10 Don rubbe gloves (6.5) us.in sapüng hads

and se at wr

'~.-

41 E 1115

12. Sarc: Scb Tecqae When Bacteri Saples AreNot Indicated .. .

12.1 Perform technque as desbe in Seon 11, exceomit 11..10. Paeli dr hads with clea pa towelafr fi ri of hads .

, .. .~~ .' '.'. '../13. Sapli Tecque L ;. 13.1 At sped sáplig ties asticay add 75 mL of

sapli solution (6.7) to glove and had to be sapled 2n

oclud glove abve wr d ~,..:~13.2 Af adg saplig sOlution, . unormy maSsgë

al su of had for i mi . . .13.3 Af massging, ascay saple the fluid of tbëglove. --: L .... ~ " ~~:':i

14. EmelOD ofBana in Sapli Solaton'. . : .;;~14.1 Enumer the ba in the saplig soluton by a

stda pla count prur suh as tht deòe inStada Metod for the Evauation of Da Prue buus soybe., di 'ag (6.9) an a suleinvarls for th anticrbi wh nec. Prsaple diutions in diution flui (6.8). Pl in dupüe::Incu plate saple at 30 :t 2°C for 48 h beore re

~ .'... . . t:. ~- ~.: .15. Deteoo of Redaeon Obt .. . ..C..:15.1 Dee at ea saplg inte ch frm

bae COUDts obted wi te ma. .; -15.2 For a mor re ap of the acvi of

pruc al raw da should be conve to common (b10) logth Reucons should be cacu fr thaver of the logth Th wi al fatate stcaanys of da if.de '. . .. t.:: ;.1

.'~.', .':. ' .i.~~...:

Ui. Compa or Tes Mater Wit a Col Mate16.1 It may be dele to compa the te m. with

a contrl mate If th is the ca an equivaent nimbeof pael should be ased to the contrl pruct on aradom ba Al te paet wi be equivat for bothpruc aloug the sc prur for an eslipruct may be diert. Bo pruc shoul be ruconcuy. Ideti of pruc us by pael shouldbe blide fr thos countig pl an anyz da Asu potive cotrl is a suca scb forulonapved by the U.s. Foo and Dr Admon.16.2 Compa, at ea saplig inte chge frbae counts obed wi te ma to cb

obted with contrl mate . .,

17. Pr an Bia . .:"1 7.1 A pron and bi stteent ca not be mae for

th tes metod at th tie., :...~;,~.;~:-."!is. Keyword : ":.' ::;'~!

18.1 anticrbi; effca glove juice; suca scb~ . ..' '.~ ~. I

15 If sie iD for ai is ii Ja ia sb bepeored to dce ap aeu. A su Ic is cl inPr E i 054.

261802

. E 1115 .' ~.

Th Am SO to Tes an MBl ru no po rø tI va allI ps ri as in cOlUedionwi ii it me in th st. Us 01 th SI ar 81 ad lh d8¡¡riail at th va of ii supl ri. an th ri at irhlrig of su ri .. fJ th aw ,esporribi/l. . i ~ . .

Th ør is su to re at ii ti by th ff ~ ~ an mu be ~'-r M yø en/I no re. ei r8 Of wi. You co _ ir ei fo nw at th st Of to ad stan sI be il to AS~. You co wi re C8 c: at . me at th ret8 cø. wt yo ma st. If yo telh ¥O c: ht no re . la ~ yo st me yo\' /a1O th AS Co or sr. 1916 Ræ sr. Ph PA 19103 .;

.\

803

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Appendix VIII

ASTME 1173

Evaluation of a Preoperative Ski Preparation

C) f: '),. U .;

.c~~ Designation: E 1173 - 93

.'-.

. . . ".~ . .'

Standard Test Method for ',. . . " ,. ..Evaluation of a Pre-Operative Skin Prep'aration 1

Th si is is ui th fW deOD E I 173; th Dumbe immedare foUow th deOD indi the ye ofor adptOD or. in the ca of rnoD, th ye of la reon. A Dumbe in ii ii the ye of la re Asupept epOD (.) inca an edri cl sie: th la reOD or re

1. Scpe1.1 The tes metod is desgned to derme the abty of

a pr-opetive sk prtion to reuce the redetmicrbia flora on the sk when us in a sk pr-opetiveprtion prur.

1.2 In th te metod metc unts ar us for alapplications excet for dice, in which ca inches arus and metc unts follow in panthesNOJ I-A Iaowed of micrbiolog teue is reui forthes pr1.3 This stanrd does not puon to adess all of the

safet problem. if any, associated wzth its use. It is theresponsibzlty of the user of this stand to establish appro-prate safet an health pracices an deerine the applic-bility of reguory limitations prior to use.

2. Reer Doent2.1 AS Stand'E 1054 Prce for Evauatig Invars of Anticrbi Agnts Use in Diect, Sati, Antic,or Pred Pruet

3. Sum or Tes ~ethod3. I Th te method is conduct on pael selecfrm a grup of volunte who, af refrg frm usg

topica and ora anticrobia for at lea. two weks exhibithi sk flora COUDts on the abomen and grin.

3.2 Actvity of the prepetive sk prtion is mea-su by compag microbia counts obted at vaoustie inte afr aplition of the prpeve trt-ment to sk site loc on the abmen and in the grinto counts obted frm the sae site pror to trent

. application.NOJ 2-Micrbi saple ar c: a mium of th ties

af trent aplicaon. The fi tw c: ar ma i 0 miand 30 mi po trent, an th th coon is ma DO le th4 h po trent but ~y be ma ia.

4. Sigca and Use4. I The prur should be us to te anticrbi-

contag preartions tht ar inteded to be fas-actg .and to signcatly reuce the numbe of org oninta sk.4.2 Perormance of th procur reui the knowledge

i Th te metod is UDde the jurcton of AS Co E.3S ODes an is th di rins of Sub E3S.IS OD Anii~

Ji A¡tsCiii ed apve Feb. IS. 1993. Pu Ap 1993. Ory

puli as E I 173 - 87. La prous edtion E I 173 _ 87.z ÁIUI Boo of ASTM Suds. Vol 11.04. .

of retions peg to the prteon of humsubjecJ

s. Appata". 5. i Colony Couer-Any of seer ty may be usfor exple, Qube colony COUDte.

5.2 Incatr-Any incur cable of mata atepetu of 30 = 2-C may be us .5.3 Sterizer-Any sUle st st cale of

pruci the condions of ~on.5.4 Tun (stp-lock)-e tl ca be re for hour

and miute5.5 Exmining Table-Any elevte su su as a 3

by 6-ft tale wi ni or si pag to alow thesubjec to ree.

'.:- . . ;'~

6. il aa Ma6.1 Bacerolog Pipee-IO.O and 2.-mL or l.-mLca.4 . . .. . '.' . .. .

'. 6.2 Water Dilution 'Bottles-Any stle gl COD-taer havig a iSO to 200mL ca and tit clos

. ~y be us 5 ' , " ... ".. 6.3 Scbing Cup-Stee gl cylide heit ~prxiy 2. em iDde diet of coDveent si topla on antomica ar to be sapled Usefu si ragefrm appxiy 1.5 to 4.0 em6.4 Rubber Policem be faoned in the lara-

to or puha fr mos la supply hous6.5 Tes Formuln-Wit dions for us6~6 Ster Drap or Dresng6-Use to cove tr

sk site6.7 Sqpling Solution-Dilve 0.4 g KH2PO., 10.1 g

Na2HP. and 1.0 g isphenoxylyeoxyoI' in iL of di wa. Includ in th formultion aninvar spc for th anticrbi in the te forul-tion. Adju to pH 7.8. Di in lOOmL volumes andst for 20 mi at 121-C.

6.8 Dilution FluiButteld's8 phoshate buferwater adjus to pH 7.2 and contag an anticrbi

J Se Fedal Reer, Vo146, No. 17. Jan 27, 1981.

41'die bao1 pi an avale frm mos lara su hou, Mi di bo of 160in i: 1i a ~ do an

awle fr Co Oil Co Ki Gl Co. or II Io Iasuly Ii6 A su aM is TEA NODAd Dr No. 327, fr meKe Co Hos Pr Bo MA 02101.7 Tri X-IOO. is ava fr Ro an Ha Co~ pt¡iac PA-i Il's Ph Bufer, JOI of W A.n of Ofic A.ia

C/i. Vo122 No. 625.1939.

830 264

\

4t E 1173

inctvator spc for the te formultion. (Se PrctceE 1054.)

6.9 Pialing Medium-Soybe~ diges ag.9

7. Tes and Contol Ski Sites7.1 . The sk site seeced . for us in evauatig the

effecvenes of the pre-opetive sk prartion shouldre~t boy ar tht ar common suca site andshould mclude both dr and mois sk ar The siteshould po ba popultions lae enough to alowdem0nsti0n; ofba reucton of up to 2.0 lagia cm2on dr sk site and up to 3.0 logia cm2 on mois site.

Balie popultions of at lea 4.5 logiolcm2 on wet sksite and of at lea 3.5 logiolcm2 on dr sk site arremmende A sutale dr sk ar is the abdomen, anda sutale mois ar is the ax or grin. ...

7.2 Abdomi trent ar ar to be loc with a5 by 5-in (12.7 by 12.7-c) site locte in the vicity belowthe umbilcu one either to the rit or left of the medUsi a 5 by 5-in (12.7 by 12.7-c) ste pa templathe comer of eah site ar maed as 1, 2, 3, and 4 di;

~n the sk us a ste suca sk maer. NumbegIS to be the sae for al abomi site numbe 1 is plaat the to~ com~ to the subjec's right, and numbe 2, 3, an4 ar ased m ordr clockw frm 1. Ea quat ofea site rents a diernt trent expo of either10 mi 30 mi or ~4 h. The reg four quat isus as a bae count site.

7.3 Simy, usg a 2 by 5-in (5.1 by 12.7-e) stetemplate, 2 by S-In (5.1 by 12.7-c) site ar delieate on

the iner as of both upp th with 2 in and paelto the leg cr below the grin The top comer of the sitear numbe benning on the subjec's rit la withnumber 1 and in order though 4. . ..7.3.1 Th 2 by 2-in. (5:1 by 5.1-c) ar rentdiert expo of 10 mi 30 mi or ~4 h, and thefour ar is us as a balie site.

7.3.2 The. abomi and groin site ar ilusted in Fig.1. A paeli may have two trents aplied to hiabdomen prvide th a 5 by 5-in (12.7 by 12.7 -e) site isdefied both on the right and lef side of the med Onyone trent, howeer, may be apli to the gr as the

rit and lef gr site ar to reve the sae trent.

8. Prur8.1 Ret heathy adult hum volunte who have no

clca evdece of detos op wounds or oter skdird whch may afec the intety of the sty and insucient numbe such tht 12 quaed abomi siteand 12 quaed grin site ar avale for trtment. Thmay reui enterig more th si volunters into thestdy, as in al probailty not everyone enter into the

stdy wi exhbit in the desgnted ski sites the bacterialpopultion reui to demonste the log reucton sp-fied in 7.1.8.2 Inct the volunters to avoid conta with anti-

crbi (other th the te formulon) for the durtion of

9 u.s. Ph XX: Unite Swe PIpe CoDvetiOD. IDCRo MD. se Qi entitled -Mibi Lits Tes" 1985.

.y.............."

",.. . ",. .....,. .'" '

u. ~ _

'\"A

.- : - :fk;'~~

-. "!i". ~

i ~ i. ;." .- --

/1.

FI 1 ii of Ap LO Of Tea Ar- :..~ . ".... . - :...: -

the, te !h reCton iii~iii . ahti~bia.:nt3antipets derats. shpo lotions baso and boy powde' V ølurte al ar to refr frmweg clothes tht have be tr wi a fac softer.Bag in bioc tr pols hot tubs sp and so forshould be avoide. .

8.2.1 Prvide volunte wi a ki of non-aticrbipena ca pruc . f~. exclusve us du the teVOlunte ar not to show or tu bae in the 24- peodpror to the aplication of tè ma or micrbi sa-plig. Spong ba may be taen' but th sk si to beus in the st ar to be exud . ,'~ - :.. '

821 If th sk si to be us includ ar th woulreui shvig pror to su, for exple, the ax thessite shoul be .shve no late th 48 h pror to the

aplicaon of te fomulon or micrbi sapÜD '..8.3 Af volunte have reed frm us anticr

bi for at lea 2 wee obta an este of baeba popuon frm a 2 by 2-i (S.l by 5.1-c) ar ofea 5 by 5-i (l~7 by 12.7-e) ab.m si and frm a2 by 2-in (5.1 by 5.1-e) ar of th lef and rit gr si~llec th saples at lea 72 h pror to ente suecmto th st. Sapli and enumeron teue desc in Seons 9 and 10 shoul be us. 8.4 Ba on th iDti este of bae bapoulon, seec suec wi hi ab si counts .

. an hi grin cots" A to of 12 gr si an 12abmi si ar reui .NOT 3-A gi trent is to be ap to oi OD ab

si OD a gi su

8.5 A send balie saple is' to be collec the daytrtment is applied. A sigle saple is obtaed frm one 2by 2-in. (5.1 by 5.1-c) site in one grin for th baliees and frm a 2 by 2-In (5.1 by 5.1-e) site withea abdmi ar8.6 rrea A.ppiicion Procedure-Imedy af

tag the send bae saple, the trent is apli~~ to la dions or as st in the prpodions On the abmen, the enti 5 by 5-in (12.7 by12.7-c) ar tht encompa four 2 by 2-in. (5.1 by5.1-e) site is prpe On the grin the 2 by S-in (5.1 by

265831

12.7-c) ar on each thgh tht encompa four 2 by 2.in.(5.1 by 5.1-c) site is pre8.7 Treaime Assignmen an Sampling Schedule-Ac-

cordig to a preed radomition, a saple of theprpe ar is taen frm the approprte site quats at10 mi 30 mi and ~4 h po trtment usg the scb cuptehnque (9.1).

NOT 4-Be th tie of trent apücation and fisapli subjec shoul avoid acvities or potions tht would causuntr sk si to CODta tr si or clot To alow thesuje soe de of mobity be th tie of trent and fisali the tr sk ar shoul be dr looy with a steDOu-ousve dr (6.6). Tl i: is apüed in su a maeras to pr th tr sk si fr cota wi untr sk.9. Microbiologca Method

9.1 Qutitative cu ar obted by the deentscb cup tehnque.10 Hold a ste scbbi cup (6.3)fiy, to the sk Ascay pipe 2 mL of ste sapligsoluton (6.6) into the scbbi cup and rob the sk wi aste robber police (6.4) for 1 mi usg modertepr. As the wa fluid an pla in a ste tetUbe. Pl a send 2.mL aluot of sapli solution inth scb cu and ru the sk ag for 1 mi with therobbe policema. Pool the two waes and enumerte thebaio. Eaaiertion of Baer in Sapli Solaton;')0.1 Enumerte th ba in the saplig solution by aStda pla count prur such as th debe inSud Method for the Evaluaion of Dairy Produc, i Ibut Us soybe-c di ag (6.9) ,and a sutale~~;,,,. .': . -' ;."....ii':. ._~. 10 Wi P ~ aD Kl A. M. . A New Met for !b QulitivelIon of Cin Ba" JfJ 01 11I~ Dmolog, Vol46. pp 198-S03. ,. ii Sld Mmulor ti ÛI of Dt Pr Qa StPl . Co Me 14th cd Am Pu Hc Asa, Iiy.as DC 1978.",.. ......N .....

o E 1173

~......~

invator for the anticrobia wher nec. Prsaple diutions in diution fluid (6.8). Plate in duplicate. ;Incubate plate saples from. the abdomen at 30- ~ 2-e, and

thos for the grID or ax at 35 :: 2-e for 48 to 72 h beforereg. , ...., ,:r . '\11. Deermtion of Reducton Obtaed '.." ... . - '. ... .~""1 1.1 Dee chages frm balie counts obtedwi the tes mate at ea saplig inte for eaanomica site. .. . .. ": .'~, ~ ~. ~~

i 1.2 For a more rec app of the acvity' ófpruct al raw data should be conver to common.cb10) log Reducton should be cacute frm

theave of the logth Th wi al fatate stcaanys of data ifde . . . ',','o~".. .. ......,12. Compan of Tes Mater With Col Mas)

12.1 It may be dele to compa the te ma wia pla or potive contrl ma or bo If so, thnumbe of te subjec should be incr The numbe ofex sujec reui wi dend upn th nWDbe"'ofcontrl po-trent saplig inte chos and tIlev of stca signcace de for the te æsIdeti of te and contrl mate asgnents shoul beblide fr thos counti pla and anyz da ,.;~12.2 A su potive contrl ma is a pr-o~

atve sk prtion apve by the U.s. Foo and i;Admtion. . A plabo forulon may be the tema without the anticrbi inent. . . ' .":' U123 Compa at ea saplig inte change ~

bae èounts obted wi the te ma to clobted wi the contrl mate . . ':,. : ,.

a ...; j~~: ;~113. Proa an Bia . ':.:i ':7~13.1 A pron and bi stteent cåot be ma forth te metod at th tie. . . .:';T:t¡

14. Keywrd ''; : "'.~lA. i anticrbia effca pr-opeve; sk

. .j,

.:;; -: ~ .~ . T1 Am So fo Tes ii Ml ti no po f8 th 'l d an ,. ii -i in CIWI an It mete in tI ~. /b d ti 8t .. ei 8d ,. dIiiiil8 d th 't d an 4Upå ri ii th ri d ~ d 4U ri .. en th aw ta. ..jf'~~:. , . ,.. ~Tl ~ Ia au 10 1I8I an ti by th re ti c: ii nU be I8 ~ ft ~ an

. II no fW, tI NION crWiIW. Yil coøi.. it tJ fo II d th ai cr fo it st. II sh be ad F i d 10 AS ~ Yii co&1 wil' QI ClII ., . me d th ralSJ8 celri wt ra nI -.. II ra ,. ,. ya CC h8 no fe . ft he ra sh ma ~':';: .. lI Ia to th Al CG on~. 1916 Ra St. PN PA 19103

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26 fj'832

Appendix ix

ASTME 1174

Evaluation of Healthcare Personnel Handwash

') ~.... D ,

~~ Designation: E 1174 (0

~ 0.~

....oi .. '. ..Standard Test Method forEvaluation of Health Care Persoruïêi Handwash Formulation 1

Th sida is ised unde the fi degntion E i 174: the numbe immedldy folowng th degnon indicate th ye of. orina adption or. in the ca of reon. the yea of la ~on. A numbe in path indica the ye of la reva. A

sui:in eplon (.) indicate an edtori ch since the la reon or miva

1. Scpe ,1.1 Th tes method is desgned to determne the abilty

of an antimicrbial hadwasg agent to give reucton oftrent microbia flora (contanats) when us in ahad wag prour. .

1.2 A knowlede of micrbiologica technques is reui

for thes prour1.3 This standd may involve haardous maeral, oper.

ations. an equipment. This stand does not puort toadress all of the safety problem associaied with its use. It isthe responsibilty of the user of this stndard to esablishappropriaie safety an health praaices an detenine theapplicaility of reguaiory /imitaiions prior to use. For morespc prutiona sttements se Note i and 2.2. Refernce Doent

2.1 ASM Stand:E 1054 Prce for Evaluatig Inactvators of Anticrbi Agents Use in Diect, Satier, Antitic,or Prrved Produet

3. Sum of Test Method3.1 Th te method is conduct on a grup of volunte

pael who have refrned from usng topica anticro-bia for at lea one week prior to the initition of the teActvity is mea by compag the numbe of a maerbacteri reovere from arciy contaated hads aferus of the hadwag formultion to the numbe reov-er frm contated unwaed hads. A broth cultu ofSe"aiia macescen, a spes of ba which produce are pigment color on an ag su is us as thecontaminant ba The acvity of the formultion .ismea followig i, 3, 5, and ~ hadwags. .4. Sigc:ce aDd Use

4.1 The prur should be us to tes the dcgeffecvenes of anticrbi had wang agents us byheath ca persriel, tht ar intende for fruent us andtht ar intended to reuce the level of contanátion

acui though contact with contate objec orpeple.4.2 Performance of th procure requies the knowledge

of retions pertng to the protecton of hum sub-jec.3

i Th te metod is unde the jurc:on of AS Committ E-35 on

Peci an is the di rensbity of Subcmmit E35.15 AnticrbiAgetsCiit edon apve July 31. 1987. Publi Sebe 1987.2 Ánnll Book of ASM Suds. Vol J 1.04,3 Se Federal Raisier. Vol

46. No. 17. Jan. 27, 1981.

;' .:0,.. 'T: , ...5. Appatu

5.1 Colony Couter-Any of severa ty may be usfor exaple, Quebe Colony Counter.

'5.2 Inraior-Any incubator capable of mantag atepe of 2S.:% TC may be us Th tempetu isreui to as pigment producton by the Seratia...:' 5.3 Sterlizer-Any sutable st str capable ofprucig the conditions of sttion is actale.

5.4 Time (Stop-lock)-e that ca rere for miuteand sends' .. .~.o., . .

5.5 Hanwashing Sink":Asi of sufcient si to pe.mit paeli to wa wiout touchig hads to si suorothcr.paeli .'.. ~ ~:

5.5.1 Wat faces) to be locte abve the sink at aheight which pets the hads to be held higher th the

~bow durg the wag prour..5.6 Tap Waier Tempeate Regor an Temperature

Monitor-To monitor and re water tempetu of 40..:%2.C. .,. . ... ."' ~. : ~- .

6. Maer aDd Reaen . .:. .6:1 .:Baaerologca Pipees-lO.O and 2.2-mL or 1.-mLcapaci." . .... . :~-~... .

6.2 Waier Dilution Boules-Any strible g1 con-taer havig a 150-200 mL caty and tight clos maybe us'. '.6.3 Erlenmeyer Flask-2-L capaty for cuturg teorg. ..; - oJ . .~ ..' ¿ . J .'.:. 6.4 Baseline Conrol Soa, a liuid cae sop or other

. liquid sop contanig no antimicrbi6.5 Tes Formulion-Dions for us of tes formul-

tionshould be include . if avale. .Jf ther ar not any

avale, us dions prvide in th te metod (se9.5). .. :.. . ~; ,,',:0:': /:.,.. .: ':

. ~ ~6.6 Gloves-Lofittng glove. of late uned pa: seg non:.anticrbi prpertes 6:: 6.7 Sampling Solution 7-Dilve 0.4 g KH2P04, 10.1 gNa2HP4 and La g islphenoxyplyetoxyetnoi8 in

.~; . ... .' . .

e. Prdile baologca pipees ar avale frm mos loc

Iara supply houS Mi diuton bottes of l60mL caty having a SC-cp clos ar

avale frm Comig Gla Co.. Kible Gla Co. or mos loc latorysuly hous. 6 A sue glove PI 8873C (sie) Aeu La Prur Glovefrm Amer Ph Ub GIe CA 9120. A zo ofinhibition te su as AATCC Tes Mei 9(1965 may be us to evuamtcrbi pr of gl .uTCC Tøi MøJod. Amerca Ason ofTex Ch an Colo 1968 Tedca Maua Seon B-17.7 Peii A. F ~ '" Miol of th Ha Evaua th Efec ofSiuca Sc - ~/o il JlIal Micioog. Vol 14, pp. 125130.1973

· Trion X.ioa, Robm and Ha Co~ Phphi PA.

833268

,I-L died water. Adjus pH to 7.8 with 0.1 NHQ or 0.1 NNaOH. Dins in 75.mL volumes and ster for io mi. iii.c.6.8 Di/uiion Fluid-Butterfelds ste phosphate buf.

er waier9 adjus to pH 7.2 with sutable invator forthe anticrobia. Adjus pH with O. I N HQ or O. i NNaOH. (Se Tes Method E 1054.)

6.9 Agar-Conta Soybe-c diges aglO plussutable inctvator.

6.10 Broth-Soybe-cfl7. Test Orm

7.1 Seratia mtcescen ATCC No. 14756 is to be us asa maker org. Th is a st havig stle pigmenta-tion.NOI 1: WUD-The aplicaon of micrrg to the sk

may ÍDvolve a hea ri Pnor to aplyi the S. mJ st toth sI th lUtibioti sevity prfi of th st shoul bede If the st is not seve to ûetayc: do not us it IflU inecon oc the lUtibiotic sevi prfi shul be maavale to th at clinician. FoUow th p; la cotatin an wa wi th teforulon, th pael's bads ar to be sati by scbbi wi a70 % etol soluton. Th pu of th alhol sc is to deany re S. rresce.

7.2 Preparation of Marke Culure Susenion-Fromstk cutu inocte Seratia mtcescen ATCC No.14756 in 2-L Oa contag 1000 mL of Soybc-cdi brth (6.10). Incuba for 24 : 4 h at is.c. Sti or"ùe the suon before eah aluot withdrwa Asysuon for numbe of org by membre

Dltrtion tehnque or su inocon at the benningand end of the us peod Do not us a suon for moreth 8 h.

8. Pali: 8. 1 Rct a sucient numbe of heathy adult hum

volunte who have no clca evdece of de opnwounds hangnail or oter sk dird tht may afec theintety of the te and such tht 12 suec complet the

. sty... 8.2 Ii the volunte to avoid conta with anti-crbi (other th the te forultion) for th duron ofthe te and for at lea one we pnor to th te Threcton include antibi~nta antipetsdeorats shpo lotions and so al su mate. as acds ba an solvents Bathing in biocde tr polshot tu sp et., should be avoi Volunte ar to be

prvided with a kit of non-aticrbi pena capruct for exclusve us durg th te an rubbe glovesto be worn when conta with anticrbi caot beavoided

9. Prure9.1 Af paeli have refred frm us anticr

bia for at lea 7 days they peorm a 30-5 prce wa in

Buu's PI Bufcr, JOI of 1M .4 of Oju: Â1yria.Ch Val 22 No. 6~. 1939.10 UII Sl Plupd XX: Uiire5i PI CoYeID Roe, MD, Ci ei ~Mi üi Tcs-

o E 1174 ~':~-:ò:":'-~~)!'''f ~. .' ""

pr quicly enoug to avoid de ba counts due tocontiue acon of the te formultion? Wh the deon. tofawe th compan of rets ac st the invesr shoulinca whet or Dot au invar ha be includ '

balie counts obtaed wi te mate12. Compan of Tes Material with a Control Mater

12.1 It may be d~îe tò compa the te mate witha control ma. If th is the ca, an equivaent number'of P.e1 should be asgned to the contrl product on a

radom ba AU te paete wi be equivaent for" '.prue aIthoug the wa prur for an esliedpruc may be diert. Both pruct should be ruconcuntly.12.2 Compa, at ea saplig inte chge frm

bae counts obted with te mate to chgesobted with control. mate.'." ,". ....\.13. Prio an Bia . ".''13.1 A Proñ and bi ~ment caot be mae forth te metod at ~ tie. . .. ,

. ';...- '.~ .....

, .

:¡: :

10. Enumeration of Baeri in Sapli Soluton .:. ..

10. i Enumerte the S. macescen in the saplig solu-tion usg stdad micrbiologca tehnques suh asmembre fiter tehnque or suac inoction tehnque.Pr saple diutions in diution fluid (6.8). Use soybe-ca dige ag with sutale invator as revermedum. Incubate pr plate 48 h at 2S : 2.e.Stada plate countig prur ar us to count onlythe re pigmente S. macescen.

i 1. Deermtion of Renctoni 1.1 Deere at eah saplig inte chages frm

Th Am So fo Tsr an Ma ta 11 po teig th ve at My Pf ri -. in C:UlIlIe:ilwi an 1_ miiutioll in tI ~. lh at th 8t .. øi 8t tt .. at th va at an u:,. ri, an th ri of Iiilrngiiment 01 &U ri. .. en th ow ~. ; ; '. 7: ..'~'.. :., ..

Th st is BU to re li an ti by th re te COlli"' an mu be If -v fN ,. ønII no nM, Di ff or wi. yOI c: ., it., fo AM at th ai or fo 6t atan sh be edre to AS He yOI c:_ wl I8 Qf col: li. ", of th tete cc. wt yo me &t. If yo fe tt yo COI.ir ,. no re . ,. he yo si II yaVÎ kn to tJAS Co6l øn St. 1916 Ra St. Pl PA 19103 ..:: . '. .:. ,_

" ....

,',:",

.-;: .

. _.. .

'. .....

.-.'. .,.-

270835

(,

Appendix X

Hand Rub Test

').. 1~ iI..

Draft Standard Test Method for:Evaluation of Alcohol-based Hand Rull Formulations.

The objective of this test is to evaluate the effcacy of alcohol-based formulationsdesigned to be used as waterless hand rubs. The study consists of a 5 - 7 day washoutperiod followed by the treatment. Subjects, who are enrolled, are instrcted to refrainfrom using antibacteriaVantimicrobial soaps, medicated lotions and creams, and/ordandrff shampoos until the study is completed. Durng the treatment period, the handsare containated with E. coli via submersion into the suspension. We propose that

subjects' hands be containated five times followed by bacterial recovery from thefingertips.

C).- "Iv , ,;.

1

Draft No.5Q~ 0/1996-

Standard Test Method forEVALUATION OF ALCOHOL-BASED HA"-i) RUB FORI\llJU nONS

1. SCOPE

1. 1 TIs test is designed to evaluate alcohol-base fonnulations for utility and

effectiveness as a hand-rub designed for repeated use in patient heath care.

Note 1 - A knowledge of microbiological technques is required for these procedures.

1.2 Antimicrobial handwashing products traditionaly have been formulated Vtith soaps

and detergents and are meant for use in reducing the transient microt1ora and the transmission of

infection. Alcohol-based fonnulations provide a product that is fast-acting and requires no water

or sink.

Contamnation of the hands by dipping into a high-count liquid suspension produces

-"" consistent levels of contamnation and reduces the number of subjects required to produce reliable

results. Six percent isopropyl alcohol is used as a standard in this test procedure. The sampling

of the hands is limited to the fingertips. However, this test design yields a structred test with a

consistent sampling technque using the portion of the hands most often involved in transmission

of microorgansms.

Other test organsms have been used in this test, for example, Sen-atia marcesens and

Staphylococcu aureus (with IR consent) to represent the most likely contaminant transferred

with the gram positive bacteria of the skin.'\

273

2

Strains such as S. marcescens and S. crúreus have been utilized because ~e~' -:..- i... ...."ily

differentiated from the background hand microflora.

1.3 Ths standard may involve hazdous materials. operations, and equipment. Ths

standard does not purport to address all of the saety problems associated with its use. It is the

responsibility of the user of this standard to establish appropriate saety and heath practces and

determe the applicability of regulatory litations prior to use. The user should consult a

reference for the laboratory safety recommendations. i

2. REFERENCED DOCUMNTS

2.1 ASTM Standard E 1054 Test Methods for Evaluating Inactivators of

Antimicrobial Agents Used in Disinfectant, Santier and Antiseptic Product.

2.2 References to technical publications are listed at the end of this document.

3. SUMMARY OF TEST METHOD

3.1 Ths test method is conducted on volunteer panelists who have refrained from

using antimicrobial for at least 5-7 days prior to the initiation of the test. At least 15 panelists are

selected for the test.

3.2 The subjec's hands are simultaneously contaminated with a high - count culture

of a specific test organism which is dried on the hands. Afer assessment of the number of

organisms applied to the hands. the test product or the control standard is used as directed by the

manufactrer in the "Directions for Use" or by a defined hand rubbing procedure with careful

attention to specific instructions for rubbing the hands. The fingertips are sampled to assess the

i CDC.NI~ i 993. Biosaety in microbiology and biomedical laboratories. 3 rd Edition. Deparent of Heath and

Huyan Services. Washington. D.C.

274

3

recovery of the number of organisms applied and the reduction in count afer i.::e :::::-~ ;--~..'lct

or the standard.

3.3 A control standard (60 percent isopropanol) is run as a control with every test

product. The reduction in count of the test organsm afer application of the test product or the

standard is calculated as the mean log reducton (Rotter, Reduction Factor or RF) of the

repetitions perfoimed. The sae subjects can be used to repea the test with a short rest period

in-between test.

4. SIGNICANCE AND USE

4.1 The procedure described in this test method can be used to evaluate the

effectiveness of an alcohol-based hand-rub product in reducing the trasient ITcroflora of the

hands. A comparson with a known standard of 60 percent isopropyl alcohol in each test provides

a reliable estimation of effectiveness based on the number of repetitions and the use of a control

product. (1,2)

5. APPARTUS

5. 1 Colony Counter - Any of several tyes may be used. for example, a Quebec

Colony Counter.

5.2 Incubator - Any incubator capable of maintaining a temperature of35 :: 2C may be

used.

5.3 . Steriizer - Any suitable steam-sterilizer capable of producing the conditions of

sterility is acceptable.

5.4 Timer - (stop-clock) that can be read for minutes and seconds.'\

275

4

6. MATERILS AND REAGENTS.

6.1 Petri Dishes - 100 by 15mm required for performng a standard plate count. 2

, .,0._ Bacteriological Pipets. 2.2 or 1.1 ml capacity"

6.3 Suitable dilution blans with a capacity to permt at)propriate diution.

6.4 Control Standard Product - 60 percent isopropyl alcohoL. Use instructions will be

3 nù for 30 seconds, rubbing in the sae maner as the test formulation. If there are no

instructions, a stdardized pattern of hand rubbing should be use.

6.5 Test Formulations - Directions for use should be included.

6.6 Sampling Solution - Trytic soy broth is the solution used as the saplig fluid (10

m1). One Petri plate base is used for each hand for estimation of bacteria released afer

contamination and treatment.

6.7 Dilution Fluid - Butterfeld's phosphate buffered water adjusted to pH 7.2,

.icontaining an antibacterial inactivator specc for the active ingredients in the formulation. An

Inactivator specifc for the antimcrobials in use in addition to alcohol in a test formulation should

be added to the dilution fluid. Alcohol is neutraized by dilution.

6.8 Test Organisms - Microbiologic cultures of the selected test organsms. Although

Escherichia coli ATCC i 1229 is the test organsm used historically by Rotter (4) other simulant

or marker organsms can be used. Cultures should be transferred several days in succession

working stepwise from a ten-nu test tube to larger volumes of inoculum. A two-liter volume has

Presterilized/dispsable plastic petr dishes are available from most loc laboratory houss.) Presterilized/dispsable bacteriologica pipets are available from most IOQI laboratory houses.

; Butterfeld's Phosphate buffer. Journ of the Asation of Ofcial Agrculrul Chemist Vol 27. 1939.

276

been used to immerse the hands. but this can be vared to accommodate the req.~:;e::,:-.~ -&'.ie

test. The culture used for contamination of the hands must be 18-24 hr old when used. If more

~¡;a.-¡ ::mc series of tests is done in one day, a second culture should be prepared for the second

series so that the age of the culture is correc. Cultures should be grown in trytic soy broth.

6.9 Erlenmeyer Flasks (4-liter capacity) for preparation oflarge volumes of culture for

hand contamation.

6.10 Sterile contaers for imersion of hads for contamnation.

6.11 Trytic soy agar and trytic soy agar with 0.05 percent deoxycholate added

specifcally to enumerate E. coli 'and to inhbit the normal staphylococci.

6.12 Trytic soy broth.

6.13 Test fòrmulations. Directions for use should be included. If they are not, the

material may be tesed using the same instructons used for the control standard.

6.14 Bland non-antimicrobial hand soap (liquid) such as BabySan. S

6.15 Control standard product - 60 percent isopropanol. (v/v)

7. TEST PRODUcrS

7.1 Fifteen panelists shall consist of healthy adult volunteers. They shall not have

taken antibiotics or used topical antimicrobial products for one week prior to enrollment in the

study. Panelists shall not use topical products containing antimicrobial (shampoos. antiperspirants

and soaps) or expose their hands to harsh acids or bases for 3 days prior to the test. They shall

not have skin wounds, lesions, abrasions or dermatitis. The number of panelists historically used

~

Product of Huntingion Liboratorics. Huntington IN.

'J'" "'~ , ,

ô

for tlus test has been 12- i 5. (3.4.5. 6,) Calcuiations of variance have sho'NT th",. :: :.: .:::~ .Jie

number at a signcance level ofO.OS based on the level of contamination. It is prudent when

Jcäl.i'5 with human subjects to include some extra ones. Ths may avoiè expensive repetition .and

at worst improves the statistical basis.

8. EXPERINTAL DESIGN

8.1 Afer washig briefly with a mild liquid non-antiITcrobial soap, al of the seleced

panelists contaate both hands with the seleced tes organsm. Evaluation of the

contamnation level is made, the subjecs hands are rubbed with the control product accordig to

directions and the ITcrobial count evaluated. The same contamnation is repeated, the level

sampled and is followed with the test fonnulation and then evaluated for microbial reduction. At

leas three repetitions oftrus procedure (up to five have been routinely used) using the contra!

product and the test product is recommend~.

9. PROCEDUR

9. 1 Before beginning the test, each subject thorougly scrubs his hands for 2 nuutes

with a bland, non antimcrobial soap (such as BabySan) and driesthorougly with paper towel.

9.2 The hands are immersed into the 18-24 hour culture of E. coli ATCC 11229 or

other selected test organsms to the middle of the palms for S seconds. The hands are removed

and allowed to carefully drain and air dried horizontally for 3 minutes with continued pronation

and supination to prevent pooling.

9.3 The release of surviving contamination from the fingertips is measured by rubbing

and kneading the fingertips for 60 seconds against the base of two 9-cm Petri dishes (one for'I

').. 8Iw i

7

each hand) containing 10 ml of sampling fluid with specific neutralizers. This fl;.;: :: ::~''': - ~

plated and incubated at 35 = 2C.

9.4 Imediately following this sapling, apply the control standard product using 3 ml

poured into a cupped hand and rub thoroughy, covering all pans of the hands. Rub the product

into the hands for 30 seconds. Repeat the process for another 30 seconds.

9.5 Afer recontanation of the hands, the sae sapling procedure (9.3) is executed

and is imediately followed by the tes disinecant using the sae test conditions as for the

control standard product with care to cover the thumbs, nails, ungual and sub-ungual areas,

fingers and crevices.

9.6 Application of the disinfectant is followed by kneading in the 3apling solution.

Viable counts per millliter are determined by dilution and plating on tryptic soy agar. Dilution

fluid should contai specc neutralizers for antimicrobials other than alcohoL.

9.7 A diagraratic representation of the procedure foUows:

'\

c)., 9.. .

3

9.8 The counts per ml are determined and recorded, converted to logio, and the

JilTi.í':l1Ce in count from the pre-disinfection enumeration calculated as log reduction (Roner.

Reduction Factor). Mea log values from the left and right hands of each subjec are calcuated

pre-use and post-use of the test substance and the stdard.

9.9 Several statistical comparsons ca be made. Comparson of the mean log

reducton betwee the product and the stdad using a Wilcoxon matched signed ra tes a

one sided test of signcance (p0. I) is recommended in the Austan and Gennan Guidelies ( 1).

At-test comparson of means and a mixed model analysis of varance (ANOVA) have been used.

9.10 If more than one product is tested during a day's tes it is only necessa to

perfonn the test on one saple of standard (60 percent is isopropanol).

10. PRECIION AND BIAS

10.1 A precision and bias statement ca nOt be made for this test method at this tie.

1 1. KEY WORDS: Rotter, Vienna Model, hygienic hand disinfection, ski microt1ora hand

rub, alcohol, inection trannussion.

"I

('.

280

'f

REITRE:'CES

:. ..\~strian Society for Hygiene, Microbiology and Preventive Medicine (Guidelines Nov -i,1960). 1981. Evaluation of the disinfecing power of procedures for surgical handdisinfection. Osterr. Kranenhauszig. 22: 32 and Hyg. and Med. 6: 10.

2. Koller, W., Rotter, M. and Kundi, \1. 1978. Evaluation of procedures for hygienic

disinfection of hands: Comparson of two methods for arifcially contamnating hands and useof an automatic colony counter. Zbl. Bak. Hyg. I Abt. Orig. B. 107: 38-47.

3. Maner, P., Rotter, M. and Mittermayer, H. 1975. Compartive investigation of recoveringbacteria from arifcially contamnated hands. Zbl. Bak. Hyg. I Abt. Orig. B. 160: 412-431.

4. Rotter, M. 1984. Hygienic hand disinfeeton. lnfee. Control. 5: 18-22.

5. Rotter, M., Koller, Wand Wewalka. G. 1980. Povidone-iodine and chlorhexidine gluconate-

containing detergents for disinection of hands. 1. Hosp. Wec. 1: 149-156.

6. Rotter, M., We.lUalKaJ G and Koller, W. 1982. Infuence of some varables on the results ofevaluations of procedures for hygienic and disinfection. Hyg. and Med. 7: 157 -166.

"\

C) 81.. ..

Appendix XI

Statistical Proposal

'\ 8". ,hi .,.

Statistical Proposal

The 1994 Monograph proposes a statistical approach for in vivo testing of SurgicalScrubs (§ 333.470(a)(2)(b)(I)(iii)(F)), Healthcare Personnel Handwashes (§333.470(a)(2)(b)(2)(iii)(F), and Preoperative preparations (§ 333.470 (a)(2)(b)(3)(iii)(G).Ths approach is based on comparson testing of predicate products to assess testformulation effcacy. We find ths approach to be financially burdensome and

unecessar to demonstrate product effectiveness. We have also proposed many

additional types of in vivo studies which are listed in Tables I-VI; these will requirestatistical evaluation as well.

To achieve statistically significant results with antimicrobial studies, we reviewed theFDA monograph for antiperspirant drg products. That monograph requires that in orderto label a product as an antiperspirant the effcacy testing of the product must demonstrateat least a 20% reduction in perspiration. Paraphrasing the monograph:

The hypothesis that reduction in perspiration exceeds 20 percent is testedstatistically using parametrc or non-parametrc technques. The nullhypothesis is as follows: (Ho: the median percent reduction is less than orequal to 20 percent), the alternative hypothesis states: (Ha: the medianpercent reduction is greater than 20 percent). The null hypothesis (Ho:) istested against a one-sided alternative, Ha:, at a predetermined level ofsignificance, usually taen to be 0.05. Rejection of the null hypothesis at

significance level 0.05 will justify the conclusion that at least 50 percent ofthe test population will obtain a sweat reduction of at least 20%.

Therefore, we propose, for the in vivo antimicrobial testing outlined in Tables I-VI foreach category, a binomial criterion be used as follows

The binomial test with one-sided significance level will be used to test the hypothesis thatless than or equa to one-half of the subjects achieved an "XYZ" log reduction versus thealternative that more than half achieved an "XYZ" reduction. Rejection of the nullhypothesis at significance level 0.05 will justify the conclusion that at least 50% of thetest population will obtain a log reduction of at least "XYZ."

Utilizing the binomial test to show that a product achieved a target level of performancewould assure that at least 50% of the test population would achieve the required

reduction. As compared to the statistics suggested in the 1994 TFM for in vivo testing,the binomial criterion would assure that better products make it to the marketplace. It isinherent with the binomial approach that the mean level of activity for test products be

C) 8 ':hi .J

greater than the target level, and that greater than 50% of the test subjects achieve activitylevels greater than the target level in Tables I-VI in order to achieve statisticalsignificance. Ths is a more strngent requirement than the proposed monograph. The1994 monograph requires that formulations achieve a target level of activity on averageand achieve a party position with a curently accepted predicate product. The partyposition allows for products to differ in mean activity by as much as 20% from thecontrol product and stil meet the monograph requirements. Therefore, the test productcould be allowed to be 20% less effective than the predicate. An example of eachapproach follows:

Party Criterion (1994 TFM)

If the predicate product achieved a 1.3 log reduction, a test product couldpotentially achieve a 1.04 log reduction and stil be at party with the

predicate. With ths approach, there is a potential that less than 50% ofthe test subjects would have achieved a 1.0 log reduction. (The lowerlimit of the 95% confdence interval about the mean would be much lessthan 1.0 log.)

Binomial Criterion

The binomial proposal with one-sided signficance level will be used totest the hypothesis that less than or equal to one-half of the subjects

achieved an "XYZ" log reduction versus the alternative that more thanhalf achieved an "XYZ" reduction. Rejection of the null hypothesis atsignificance level 0.05 will justify the conclusion that at least 50% of thetaget population will obtan a log reduction of at least "XYZ".

No. achieving:; XYZ reductionTota No. in Panel needed for statistical si~nificance

30 2040 2650 32The binomial approach of requiring specific levels of activity to be statistically metreduces the financial burden by simply requiring that they show effcacy of their product.The large panels of subjects required to meet the party position would be an ineffectiveuse of test subjects in the research and development process. The binomial criterionallows for a reduction of test subjects. When combined with the log reduction criteria

C) R ~..u -

identified in the tables, a more effective product wil result using this approach, ratherthan the party statistics as proposed in the i 994 TFM.

') 0 ~..0:"

Evaluation of the Skin DisinfectingActivity and Cumulative Effect ofChlorhexidine and Triclosan HandwashPreparations on Hands ArtificiallyContaminated with Serratia marcescensC.A. Sartzokas, MD; J.E. Corkill; MSc. FIMLS; T. Makin, SA, FIMLS

ABSTRACTThe initial and cumulative effcacy of two antiseptic

hand wash preparations in eliminating Serratia mar.cescens from hands was evaluated on volunteers. Twoantiseptics with persistent skin antibacterial activity,4% chlorhexidine gluconate in detergent and 1.5% trioclosan in natural soap, were studied in a new protocoldesigned according to Food and Drug Administrationguidelines. After a single handwash, both preparationsexhibited a degerming action statistically superior tothe mechanical elimination of the marker organism thatwas achieved by the nonmedicated controls. Followinga further nine hand recontamination sequence with 109

colony-forming units (cfu)/mL S marcescens (mean pre-

disinfection baseline, (ogio 6.6), the efficacy of chlor-hexidine and trclosan was significantly augmented: themean logio reduction factors were 4.15 and 3.78, respec-tively. In the absence of internationally accepted testingstandards for antiseptic hand wash products, the signifi- .cance of protocol variables is discussed. The advan-tages to preventative microbiology of antiseptics withpersistent skin antibacterial activity are highlighted.

(Infect Control 1987; 8(4):163-167.j

INTRODUCTIONl1ie potential value of antiseptic handwah products in

hospital hygiene is primarily based on their degermingactivity following a single application. Following multipleapplications, however. the deposition of active anti-microbial on the skin may lead to enhanced antimicrobialeffcacy. The significance of such a remanent (persistent)activity has been recognized and is indeed specified by the

Frmn 11i~ lhlill(Tsity D~parl",ml of ,\(~dical Microbiology, Ruval Liv~rpoolHospitnl, LilJ~rpool, Uniud Kinirdom.

A.tlrlrm r~prini r~qu~s/. 10 C.A.. Bartznhos, MD, UnilJ~rsit~ D~pnrlmmi ofCliiiical Jlicrobiology. Clatl~rbnrlg~ HOJpital, ß,bing/on. Wirral, L63 .tJY,l':nitrrl Ki7lgrlom.

INFECTION CONTROL 198í/\'ol. 8. .Vo -I

Food and Drug Administration (FDA). i The FDA defines

a health care personnel handwah (known in Europe ashygienic hand disinfectant) as "a non-irritating anti-microbial-containing preparation designed for frequentuse; it reduces the number of transient microorganismson intact skin to an initial baseline level after adequate

wahing, rinsing, and drying; and it is broad-spectrum,fast-acting, and, if possible, persistent." Such persistentantimicrobial activity remaining on the skin has beenshown for hexachlorophene, chlorhexidine, and tri-c1osan, but not for the iodophors or alcohols.2-8

In Europe, the evluation of antiseptic handwash prep-arations is based on the elimination of a marker organismfrom the hands, usually Escherichia coli, following a singleapplícation.9.io In the United States, Serratia marcesce1l ispreferred and the effect of repeated hand washings istaken into consideration. i Within the FDA guidelines, wehave developed a convenient test protocol and deter-mined the initial and cumulative effcacy of chlorhex-idine- and triclosan-containing handwah preparations ineliminating S marcescens from artificially contaminatedhands.

MATERIALS AND METHODSMaterials. Two antiseptic hand wah formulations were tested: 4%chlorhexidine gluconate in a nonionic detergent base

(Hibiscrub~ iei Pharmaceuticals pic), and 1.5% weight-to-volume ratio tric10san (Irgasan~ DP 300. Ciba-GeigyAG) in a natural liquid potassium soap, pH 9,

Since no direct base for Hibiscrub was made available by

the manufacturer, a liquid Castile baby soap with olive oil(Baby-San~ Huntington Laboratories. USA) was used as acontrol preparation for the chlorhexidine gluconate. A

liquid soap base. identical to Irgasan. but without tri-closan or any antimicrobial preservatives was used as the

second control preparation.A nonmedicated natural bar soap. without anti-

microbial preservatives (Simple~ soap. The Albion SoapCompany Ltd) served as an initial hand cleanser.

163

The bacterial inoculum consisted of a 72-hour-oldculture (2 L) of Serratia marcescens ATCC 133880 in Tryp-tone Soya Broth (Oxoid, code CM129) containing circa109 colony-forming units (cfu)/mL. The bacterial suspen-sion was agitated immediatelybefore each application andwas not used for longer than six hours.

Sterile 0.075 M phosphate buffer (pH 8) containing0.1% Triton X-IOO, 0.3% sodium thiosulphate, 3%lecithin, and 10% Tween 80 was used as a deactivator.Preliminary tests showed that this composition effectivelyquenched the in vitro bactericidal activity of both chlor-hexidine gluconate- and triclosan-containing test antisep~tic formulations, against S marcescens.

Sterile tryptone water (Oxoid, code CM87) coniaining0.3% lecithin, 0.3% sodium thiosulphate, and 3% Tween80 was used as diluent. The recovery medium was Mac-Conkey Agar without salt (Oxoid, code CM7b) con-taining 0.3% lecithin and 3% Tween 80.

Twenty-four volunteers were tested. All had short tonormal length fingernails, and exhibited no visible skininjuries, eczema, or apparent skin disease. During testingno rings or wrist watches were worn. One week before theassessment, each wa supplied with nonmedicated bars ofsoap and sachets of whole body cleanser and instructednot to use any toiletries (eg. medicated soaps, deodorants,antidandruff shampoos) which may contain antibacterialagents, or handle household disinfectants. They were alsoasked to refrain from swimming in chlorinated pools.

Washing and rinsing were conducted under runninglukewrm tap water: pH 7.4, total chlorine -:o.i ppm,hardness 92.1 mg/L CaC03, average colony count -:3 cfu/mL. Sterile papertowels (Kleenex4P) were used for drying.

'MethodsAs an initial hand wah, all volunteers wahed their

hands with the nonmedicated bar soap for 30 seconds,rinsed for 30 seconds, and dried.

The volunteers' hands were anificially contaminatedwith a total of 5 mL (in two aliquots, each 2.5 mL) of thebacterial suspension. The bacterial inoculum wa pouredonto cupped hands and volunteers rubbed over theirhands, not above the wrists, for 45 seconds. The inoculumwa allowed to air-dry for one minute, while hands wereheld away from the body. All subsequent hand con-taminations were similarly performed.

To establish a predisinfection baseline, the hands werebacteriologically sampled immediately after the artificialcontamination as follows: sterile loose-fitting "gloves"(polyethylene bags, 15 cm x 38 cm), each containing 75mL of stripping fluid, were donned simultaneously by twoattendants and occluded above the wrist. The hands weremassaged in a standard manner. One minute later the"gloves" were removed and their contents pooled. Thehands were rinsed and dried.

The pooled stripping fluid was serially diluted and 0.1mL aliquots of neat and 1:10: 1:100: 1:1.000; 1:10,000: andI: 100,000 dilutions were spread over the surface of. Petridishes containing recovery medium. All cultures wereincubated at 23°C for 48 hours and th~ colony-formingunits of the marker organism, in plates containing 30 to300 cfu. were enumerated. All subsequent postdisinfec-tion samplings were similarly performed.

164

a 5Ü~co+:o:J"0~ 4ccoQ)

Er

ffff~

3

2. Triclosan

. Chlorhexidine

. Control01 I

oneI

fourI Iseven ten

number of hand washes

Figure. Serratia marcescens mean reduction factors. averagedfrom 12 volunteers. following one. four. seven. and ten hand-washings with 4% chlorhexidine gluconate- and 1.5% triclosan-containing handwash preparations.

EvaluationThe test antiseptic preparations were evluated on two

groups of 12 volunteers. Before each wah with a testpreparation the hands were always artifcially con-taminated, as described preiously. Ten consecutive wah-rinse sequences were performed using the following pro-cedures.

A 3 mL aliquot of tap water was dispensed onto cuppedhands, and volunteers rubbed over hands. Immediatelyafter application, a 3 mL aliquot of a test preparation wassimilarly applied, and the hands (including the lowerthird of the forearm) were washed energetically, in astandard manner, for 30 seconds. This procedure was

then repeated. Volunteers rinsed hands for 30 seconds

before and after each bacterial sampling.Four postdisinfection samplings were taken, after the

first, fourth, seventh, and tenth contamination-wash-rinse sequences.

Before evaluating each antiseptic hand wash formula-tion, a single artificial contamination-wash-rinse-sam-piing with the reciprocal control preparation wa per-formed. "The experimental schedule and intervals of handsamplings are diagrammatically shown in Table L.

RESULTSFor each volunteer the number of bacteria recovered

from the pooled stripping fluid obtained from sampling

Skin Disinfecting .1ctivitv/Bartzoka et al

TABLE 1EXPERIMENTAL SCHEDULE AND INTERVALS OF HAND SAMPLINGS

Schedule Contamination Wash-RinseInitial Handwash

Conirol HandwashTest Anlisepiiç Handwash

Wash 1Wash 2Wash 3Wash 4

Wash 5Wash 6Wash 7

Wash 8Wash 9Wash 10

Sampling

BaselineControl

(1)

(4)

(7

(10)

TABLE 2SERRATIA MARCESCENS MEAN REDUCTION FACTORS (RF),FOLLOWING (1), (4), (7), AND (10) HANDWASHINGS WITH CHLORHEXIDINE GLUCONATE-AND TRICLOSAN.CONTAINING HANDWASH PREPARATIONS

Mean 10910Handwashes RF ~ SO

(S) 6.63 : 0.31

(C) 2.56: 0.20

(1) 2.61 : 0.33

(4) 3.41 : 0.37

(7 3.95: 0.29

(10) 4.15: 0.34

(S) 6.66: 0.35

(C) 2.72 : 0.37

(1) 2.91 : 0.42

(4) 3.22: 0.41

(7 3.50: 0.52

(10) 3.78 :: 0.47

Test HandwashPreparationsGroup A (12 volunteers)Predisinfection baselineControl base

4% Chlorhexidinegluconate in non.anionic detergent

Group B (12 volunteers)Predisinfection baselineControl base

1.5% Triclosan in

potassium soap

Significanceof Difference

P~O.l

P~ 0.01

P ~ 0.01

P~ 0.05

P~ 0.01

P~ 0.01

P ~ 0.01

P~ 0.01

the predisinfection baseline (B); effect of the controlpreparation (C); and the degerming activity of the testantiseptic hand wash following (I), (4), (7), and (10) con-tamination-wash-rinse cydes were transformed to logiovalues. The login values obtained for each samplingperiod were subtracted from the initially determinedbaseline level and the difference expressed as a reductionfactor (RF). The mean RF and standard deviation (SD)per group of volunteers for each sampling period were

calculated.Within a volunteer group, the significance of dif-

ferences between a single application (first) of test antisep-

!.\'FfeTlON CONTROL /987/Vol. 8, No. -I

tic handwash and the reduction brought about by thecontrol preparation, as well as the differences between

single and multiple handwashings (fourth, seventh,tenth), were determined using the Wilcoxon's matchedpairs signed rank test (one-sided). The S marcescens mean

logio RF and SD following (I), (4), (7), and (10) handwash-ings with 4% chlorhexidine gluconate- and 1.5% triodosan-containing handwash preparations are presentedin Table 2 and ilustrated in the Figure.

DISCUSSIONEven after a single handwah, the antiseptic prepara-

165

TABLE 3HANDWASHING WITH CONTROLFORMULATION CONTAINING NO TRICLOSAN

Nurnber of (iog10) S msrcescensHandwashings Surviving1 3.364 3.527 3.5910 3.29

tions tested exhibited a degerming action statisticallysuperior (P 0:0.01 fortriclosan; P 0:0.1 forchlorhexidine)to the primarily mechanical elimination of the markerstrain, noted with the reciprocal nonmedicated controls.Any clinical benefits that may be conferrd by such statis-tical differences are difficult to rationalize. However, thelimitations and risks associated. with the popular beliefthat energetic hand wahing with nonmedicated soaps .isequiefficient to that achieved by antiseptics, have recently

been dispelled. i i

With repeated hand washing the effcacy of both .chlor-hexidine gluconate and triclosan hand wash preparations

was significantly augmented, despite multiple hand re-contaminations with a high challenge of S marcescens (109

cfu/mL). Similar findings with chlorhexidine-containingpreparations have been previously reported.7.12 Suchcumulative action must be attributed to depòsition ofactive antimicrobial on the skin during handwashing andretention following rinsing. In a separate experiment, theperformance of repeatedhandwahing with the controlformulation containing no triclosan demonstrated nocumulative effects (C.A. Bartokas, unpublished data)(Table 3).

The choice of S marcescens in the FDA Test Guidelines isprobably influenced by two particular features of thismicroorganism: a frequent association with nosocomialinfections in the USA, and ease of identification andcolony characterization. Furthermore, S marcescen may

be a more appropriate choice than Escherichia coli, amarker favored in European standards. since the formeris generally more resistant to chemical inactivation.Although in Europe the antimicrobial activity of chlor-hexidine and triclosan has been elicited equally satisfac-torily with other skin transients of medical significance,

an internationally acceptable standard marker remainselusive: artificially applied transients vary in their abilityto survive on the skin and, equally importnt, they mayalso differ regarding the ease of mechanical removal. 13.14

The adherence of bacterial transients on the skin and theinfluence of antiseptics on such adherence characteristicsis currntly being investigated (Bassaris Hand Lianou P,personal communication).

The FDA Test Guidelines also suggest a hand washingfrequency of at least 25 times in succession. We found thisexcessive. In our experience. the influence of energetic

handwashing on the integrity and condition of the skin.irrspective of the cleanser used. precludes more than

about ten consecutive applications. The average fre-quency of handwashing in our hospital has been

166

monitored and found not to exceed six ti;nes per workingday.15 In any case, we do not advise such a high frequencyof handwashing to our health care personnel: if handsremain physically clean after close patient. ço~tact, anantiseptic-containing alcohol rub is recommended.'purfindings, following ten consecutive hand washes, indicatethat the cumulative action of both the chlo¡-hexidine andtriclosan preparations tested could reach high values.

Peterson et ai, studying the efficacy of Hibiclens (USAequivalent of Hibisciub$), have demonstrated a potentcumulative action increasing even after 25 consecutive

handwashings.7 Though the stratum corneum acts as areservoir for topical antimicrobials,I6 we would not expecttheir cumulative activity to increase indefinitely, sincesuch handwash preparations are not used exclusively.Fears that regular usè of antiseptics with remanent skinactivity may adversely influence the composition of thenormal skin microflora are, in our view, unfounded. The"normal"Aora of the hands is dynamic and primarily. areAection of the total microenvironment.

Antiseptics with long-lasting antimicrobiai effect on theskin have been successfully applied in preoperative handand skin disinfection,17-19 routine handwashings in spe-cial care units, whole body bathing in leukemics,20 treat-ment of staphylococcal carriers,21 and, recently, perma-nent eradication of methicillin-resistant Staphylococcus

aurelL.22 In our view such antiseptics are invaluable ininfection preention and should be considered an integral

component of a balanced hospital hygiene program.However, these potent skin degerming systems should beapplied judiciously and must be regarded as a comple-ment to, but never a substitute for, asepsis and a highstandard of hygiene practice.

REFERENCESi. Department of Health, Educâuon. and Welfare. Foo and Drug Administra-

tion: OTC topical antimicrobial products. F.d.riil R.gi./.r 1978:43(4): 1210.1249.

2. van der HOCn E, Hinton N A;. An assment of the prolonged effect ofantiseptic scrubs on the bacterial flora of the hands. Ciin M.d A.Ioc) 1968:99:402-407.

3. Marpln MJ: The normal flora of the human skin.Br) D.rmoI1969; 81(supP1):2-13.

4. Müntener M, Schwarz H. Reber H: Zurchirurgischen händedesinfektion miieinem Iodophor (Betadine~ Schu'ei. M.d Wocluruchr 1972; 102:699-706.

5. Lowbury EJL. Lilly HA: Use of 4% chlorhexidine detergent soluúor(Hibiscrub) and other methods of skin disiiifection. 8r M.d) 1973; 1:510-515

6. Kundsin RB, Walicr CW: The surgical scrub. Prctical consideration. Arc!Surg 1973; 107:75-77.

7. l'icrson AF, Rosenberg A, Alatary SO: Comparative evluation of surgica

scrub preparation. SurK Gytucol Obsu/ 1978; 146:63.65.

8. Bartzokas CA, Corkil J E. Makin To et al: Assessment of the remanent antibacicrial effect of a 2% triclosan-detergent preparation on the skin.) H.yg (LDnd1983; 91:521.528.

9. Roicr M, Koller W, Kundi M, et al: Tcstmethode für die Wcnbemessung varVerfahren für die Hygienische Händedninfektion i. Teil: Beschreibung deiMethode. Z..itraJbl Biitnl MikoiJ H.yg fBJ 1977; 164:498-506.

10. Bo¡'neff J, Eggen H'J, Grun L, et al: Richtlinien für die Prüfung und Beweitung chemischer Desinfekúonsverfahren. Z.nlrt/b/8iitroi MiJrobiol H,g f8

1981; 172:534-562.11. Cox AR: In vivoAktivität von Händedesinfektionsmiuciii: Eiiie Weiiereni

wickluiig zum Haiidwah-lest-Modell an künstlich kontamiiiierten HändenHyg u M.d 1985; 10:107-111.

12. Aly R. Maibach HI: A comparison of the antimicrobial effect of 0.5% chloihcxidiiie ("ibistat") and 70% isopropyl alcohol on hands contaminaicd wiilSera/in marmct1. elin £"1' Dmna/ol 19RO: 5: 197-201.

13. Ayliffc GAl. ßabbJR. Quoraishi AH: A iesl for 'hygienic'liand ciisinfection..elin Piilhol 1978; 31 :923-928. .

14. Banzokas CA, Gibson MF. Graham R. e! al: A comparison of triclosan aii,chlor/icxidiiie preparations wiih 60% isopropyl alcohol for hygienic han.disinlectioii.) Ho.1' Inftel 1983; 4:245.255.

15. Slade PO, Wiliams E, ßanzokas CA: Psvchological coniributions to ihe con

Skin Di.iiifrcli7lg Ac/ivityIBartzoka1 et i

: :. .,

irol of ho'piial infection. in Eiscnocig N, Gla'gow 1) (cds): CUlT'��II.IlI'' iii(;ili;e,,1 P,)',h%t)', London, Gower Piess. 19B6. \'01 i.

Hi. Stoughton RI\: Hexachlorophene dcp",iiion in human 5lralum corneum.Arch Dmnoio/1966: 94:616.6.18.

17. Lill)' IIA, Lowbul"' EJL: Disinfection of thc skin with deiergeni preparationsof Irgasan Dr 300 and other antiseptics. Br M,d) 19i4: 4:372.371.

18. Eiuen HE. Riiier M A, French M LV, et al: A microbiological in.use com.parison of surgical handwashing agents.) Boii, )oiiil Surg (AmI 1979;61:103.106. .

19. Cremieux A. Guiiaud.Dauriac H. Dumenil G: Activit~ dune mousse antisep-

I.vFf.(,TION CONTROL /98';1\101. 8. ,vo. -/

tiqiie sur ta no..: noimale dcs mains. Mtdiâii, tl Malndi" .1riftelituú, 1980;1l:4.7.

20. Bodcy Gr. Rosenbaum B: Ev"hiaiion of" bacteriosiaiic soap, 1'.300, on skinnora of patients in protected environmenlS. Curr Thrr R" t 973; 15:253-260.

21. Aylirre GAJ, 8abbJR. Collns HJ.et al: Disinfection of b.iihs and b"thwaier.N,m Tim" 1975; 3:22.23.

22. Bartzokas CA. Paton JH, Gibson MF, et al: Control and eradication of meth-icillin-resistant Stophylacoccw C!urrt on a surgical unit. N Eiigl) Mtd 1981;311:1422.1425.

16ï

l1

(\ to LfV...._~:ß

\V~'"Epitrr, /'4f. (1987) 98. 337-34

¿Priritd iii OrNl Britain337

.,~,.,._Compa.ativeevaluation.(f the immediate .and sustained..,'antibacterial acton of tworegmeii", based on triclosan-and

chlorhexidine-containinghandwash preparations, on volunteers

-'By C. A. BARTZOKAS*, J. E. CORKILLt;T. MAKn.a AND E. PARRY~

*Department 0/ Microbiology. CI.attrbridge Hospital. Bebington,Wi"al £63 4J Y ; tUniversity 'Department oJ ~edica Microbiology.

Duncn Building, Royal Liverp Hospita, Pré8èo'Street. Liverpl L7 8X W ;lDepartment o/Busines Studiu, MancliterPòlytecnic, Aytn Building.Ayton Street, Manchuter.M13GH ~cS

(Acupted 30 December 1986) . "(M~Ø;Nf'

.Thedegermin eiree of a 3 mm s;~:; with'2%.trieI08, or 4% ::~'~J/e.,hexidine, in detergent and enhance efcay of either antiseic in isopropyl alcohol,was evaluate in volunters. HandwaShing with either antiseptic preparationreuce the normal flora by a factor of 10; alcohol rubbing by approximately a

" factor of ioOO.Both"regimens eliminate ¿lJ icrocus roseus, artificially inoculatedbefore every procedure. The sustained action of the same detergent preparationswas further studied in glaved and unglovèd hands by the Vinson's' finger imprint

.~ ..:tei'..In the gloved hand both antiseptlcs.inhibite Staphylous epidermidi& fori~h; In the ungloved hand however , . 'iriclosan remained active longer than-oolorhexidine. Whilst the activity of clilorhexidine wâs'short-lived against a clinical

. isolate.. of S. aureuS, particularly in the ungloved -hànd, the sustained effect oftriclosan against the same strain persiste for 4 hon èither hand.

UiTRODGCTION

:Antiseptichandwash products are extensively use in hospitals; the choice is

" often empirical.rGenerally, they are selected according to a reuction of the

'normal or trànsient handmicroflora after a single application. Following regularapplications however, certain antiseptièscan remainon-t.he"skin exhibiting sus-tained antimicrobial activity. Such remanent antimicrobial effect has been shownfor hexachlorophane, chlorhexidine and triclosan but not for the commonly useiodophors or alcohols (Peterson, Rosenberg & Alatary, 19i8; Bartzokas et al.1 983a; b).;,. A lasting remanent effectcan.enhance the .\alueof surgical and hygienic handdisinfection. Since 1978, the Food and Drug Administration (USA) recommendthat hand disinfectants possess a persistent (remanent) activity (Federal Register.

1974; 1978). Though, in theory, the significance of such antiseptic systems isrecognized, their usefulness can be impaired by the limited understanding of theadvantages which the remanent effect can confer in practice.

¡l'i! \!-

338'C.A.BARTZOKAS ASD OTHERSWe rEport experiments on the comparatin performance of antiseptic regimens.

- bas'on iriclogan-~nd.chJorhexidine~containing products; Îawhich.Jhe protocols..were' designi-to '.satisf~'- ;.thc~reuiremets=of.1lur.gi('Jand hygienic hand dis-infection.

..C'::)IATERL-\LS AXD :\IETHODS

... Experiment 1. Surgical skin disinfection .JI aterials

Test regimens. Triclosan: 2% wlv triclosan (Irgasana DP 300, Ciba-Gcigy .\G)in an anionic/ampholytic detergent bas (Aquaptli),_".fllowed by ,0-5% w/vtriclosan in 70% v Iv isopropyl alcohol BP;witli emollerïts,(Manuseptli) suppliedby Hough Hosasn& Co. ,Ltd.. Chlohezi4ir4e.:'4 % "'I\'.:ghlorhexidine gluconate(Hibitanee)ina' non-ionic detergent bas. (Hibiscruba), followed by 0'5% w/vchlorhexidine gluconate in 70% w IW isopropyl alcohol BPwith emollients(Hibisoie) siipplied. by iei Pharmaceuticals pic.. Kneading fluid. 0-(75)f phosphate buffer (pH 8), containing 0'1 % Triton X-

100, 0;3%soium-thiosulphate. 3% lecithin and 10% Tween 80.. .. DiluenLTiyptone water (Oxoid CM 87), containing 0-3 % lecithin, 0-3 % sodium-thioslphate and 3% T\\:een 80.

. "-Recer.lnium.:N~irient.agar(OxoidCM ~3), containing 0-3%,lecithin and3% Tween 80.

Volunteers. Eacliregimen was evaluate on two"separate groups of 15 hospitalstaff, allocate at random. All had short to normal length finger-nails, none had

visible skin injuries, eczema or apparent skin diseas. One week before the.asment volu,nters were supplied with non-medicate bars of soap (SimpleS, The

. Albion Soap Co. .~td.jand 'instructe not to use any toiletries (e.g.: medicate ...soaps, deodor~nts, ant~~dardruff shampoos),. which may contain.antibacterialagents, or handle laboratory, or household disinfectants. Volunters were also

asked to refrain fromß,,'i~ming in chlorinate pools. During teting no rings orwrist watches were worn. ...

Rinsing was performed under running lukewarm tap water: pH 7'4, totalchlorine C:O'lp_p.m., hardess 92'1 mgli CaCO.í, average .clony _countc:.3 per

ml.

.ilfethosInitial cleansing. V~liiIit- washed hands and lower third of forearms with

- Simple~80ap for 30 sand rinse for SO s. _ __First contt;minatio.,i.lmrnediately after (\\:ithout drying), two 2'5 ml aliquots of

.Micrococus roseus (~GTC ;523) at 108 e.f.u./ml were dispensed on to cuppedhands and volunteers rubbed over ha:nds and lower third of forearms for 45 s.Whilst the inoculum was allowed to air-dry (in c. 3 min), hands were rotateduppermost ,and fingers ~flexéd continuously to avoid droplet formation on thefinger-tips.

Baseline flra and inoculum sampling. Immediately after the inoculum air-dried,hands were placed simultaneously over two Petri dishes, each containing 10 mlkneading fluid (without deactivators), and fingers kneaded continuously for 1 min.

......--- -_."~ _....--_---- ,-,.-. - :-_~.~". --' .

r,

. Skin anti8eptics : immediate and sustained adion 339Deactivators were not added tothis kneading fluid,

since any such residues on the

skin couidpot~ntiaiiyinienere,,'ith the effcacy' of the ant isepticssu bseq uently

applied. The kneadiógèfuids from the Tight and left 'hand were p9ledand 0'1. ml.aliquots (jfserial tenfold dilutions from 100'00 '1O-1i were spread over the sunace of

'" ,:'duplicate Petri dishes containing recoverylmedium.Cultures-were incubate at" 300C..for72 h and the colony-forming units (c.f.u.) :in plates

containing 30-300

were differentially enumerated. For'each vòlunter, the c.f.u./ml recovered from': this 'and all subsequent samplings were transformed to logio values. Subsequent

Post-disinfection samplings .were similarly penormed.Seco conminaio. Hands were re-contaminateas describe above.HandUXh procedure and sampling. Immediately

after tbe,iinoculum air-dried,

a 5 ml aliquot of tap water was dispense ohto.cuppehands and voluntersru bbe over hands and lower third of forearms

for 5 s: Then a5"uil aliquot of a test

detergent preparation (i.e. AquasptS or Hibiscnib.) was'similarly dispensed:hands and forearms were washed energetically in a standard manner for 75 s. Thisproceure was then repeated. After a total of 3 min handwash, volunters rinsedhands and forearms for 30 s and towel-dried with sterile Kleenexs paper towels.Volunters kneaded fingers in fluid containing deactivators, rinsd for 308 andtowel-dried.

Third cominaion. Hands 'were re-contaminated.

,Alclwic lundru procure and sampling. Immediately after the inoculum air-

dried, a 5 ml aliquot of a test alcoholic preparation was dispensed on to cuppehands and volunters rubbed over hands and lower third of the forearms ener-getically to dryness (in c. 75-90 s). This procure was then repeated. Volunterskneaded fingers in fluid containing deactivators.

Experiment 2. Rerrnent skin antibaderieffec

Matris.T~~preparaticm. Two per cent wlv triclosan in detergent(Aquasept'l) and 4%

w/v chlorhexídirie gluconateoin detergent bas (Hibiscrub&),as in Experiment 1.Volunteers. Each preparation was separately evaluated in 12 volunteers, selecte

as previously describe. 'Firierimprint plaes. One per cent dilution of 24 h cultures of either methicilin

'and: multiply-reistant -:StaphyloU8aure1(RLH~o~:4779). or S. epidermidis'.'(KGT"~94) strains in nutrient broth (10' cLu./ml) were ,incorporate in 25 mlof-nutrient agar (Oxoid .CM-l)'&nd 80lidifiedin 121 mm; sqúare plates.

. ..

~

~'Jethods

Prdiandwash control. Before an initial 30 s hand and foréarm cleansing withSimplèli soap. the pulps of the right and left fore fingers were simultaneously

applied on the surface of two plates, overlayed with either S. aureus or S. epi-dennidis, for 30 s.

HandUXh procedure. Immediately after a5 mt aliquot of a test preparation wasdispensed on to cupped hands..Volunters rubbed o\rer hands and lower thirdofforearms energetically in a standard manner, for 75 s. This procedure was thenrepeated. After 3 min handwash, volunteers rinse hands and forearms for 30 sand towel-dried. g

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.: ~ ..

\340 , . C,A",.BARTZOK~.S AND OTHERS

J!nson '" fingtrimprint test (Vinson .et al. 1961 ),moified. .Immediately after, the',., '..."pulps of the right and left.hand foreJìngers;(f six volunters were simultaneously

. "";:llpplit'on.two'plates,overlayed'with~ther'S,'aureu"or S..epidermidis (time 0)..... . - ':d.'.~Follo",ing,30 8contact;:twoattendantsdisinfecte the ~fore 'fngers with 70%

:'~ . ',dsoprop)'lalcohol and ,donned the.non-dominantlnd with, a.glove (Dispos-a-." d f'glove- ~.'Surgik08':Ltd;rwhich waS æaledaìuid the: wrst with Sellotapee . The

dominant' hand remained unoccluded.~Volunters -rfrained -from handwashing orany contact with chemicals for 4 h. At 1,2, 3 'and 4 h after the imprint of the forefingers. the digital pulpsof:the middle"ring; Jittle.fingers and thumbs - in thatorder - were similarly imprinted and disinfected. Gloves were not removed from thenon-dominant hands, but wereampûtate around the middle phalanx of theappropriate finger. After imprinting anlClisinfection, the cû't end was sealed withSellotape*. . .

Plate were incubated at 37 OC for 24h. The zones of growth inhibition wererated 4-0 according to the criteria originàlly.a~cribe(H)y Vinson et al. (1961), asfollows ,",. Rating 'Aetivity' ,. Growth inhibition characteristics

.. Excellent "Clear ara of no grwth, with sharp periphery.

"., 3 . Goo ..., "c:;;'C1eaarea ofno:gowih with hazy periphery..:.2... '.,Fai ,. ":-,. . "'.Parialgrwth.

.' " ' -.t ' ,,"'Sligt -,,~~~ ~r.Growthalmosteqalto surrounding agar.

. .~, . o. None :. .Confluent 'grwth. equal to surrunding agar.

RESULTS AND STATISTICAL ANALYSIS

Experiment .1,Mterthe basline flora and artifical inoculum sampling. the mean logio values

. '. of M, r08eus, recovere after the tricl~sanÌ'gimen was 4.66 ; after the chlornxidineregimen, 4'68. This marker organism was never recovere from any subSequent

saniplings.The mean logio .value for the basline reident tlora (A) recovere from the 15

\'ólun'ters during the evaluation oftriclosan .was 5'88 with a standard deviation(S.D.) of 0.38. Values obtained following the A.quaspte hand wash (B) was 4'85(S.D. ~18) and the ManuSepte handrub(C) 3-0 (S.D. ~49). The A-B and A-ereuction factors wei'i-03 (S.D. ~30) and 2.79 (S.D.

(68) respecth'ely. SimilaTlY.

.the 10gl'0 values for the resident flora follo\fing. the chlorhexidine regimen (A) was',5;82 (S.D. 0.62). Values obtained following the Hibiscrub* handwash (B) was 4'i9(S.D. ~40) and the Hibisol~ handrub (C) was 2'86 (S.D. 0.55). The A-B and A-e

~ Teuctioñ'factors were::1 -03 (S.D:- ()55) and 2'96 (s.D.0'38)respecth;ely; Since'themean A-B reduction factors obtained with both regimens are virtually identicaL,a statistical test is unnecessary. Howe'ver,as the mean A-e reduction factorsappeared different, the Mann-Whitney U, test (Siegel, 1956) was applied. Therewas no statistically significant difference between the A-e reduction factors (U =83, p~ 0'(5). ..

Experiment 2

The Vinson's growth inhibition ratings (4-0) of S. epidermidis are presented inTable 1. The Aquasept* ratings obtained.at a,giventime (0-4 h) were compared

,:.:Skin,antise ptics:',~mmediate and sustained action 341. '\,,: Table ;L;"Vinson '8 finger imprint ratings oi,er4h. follouiing.lmin haruuYl.'1h with

,.:: ,_..~..,

""AqUae.pt" or H ibi.'1crub~ .'\'("lsus StaphYI9coceus-epidermidis..'

Glo\'ed hand (h I lnglovt' hand (h)\. ."--. -,

,~ f ,. - ¡\'01. 0 , 2 ,3 , .. 0 I - :2 .., 3 ..

, ''.(a) .-quasept- .

i 4 4 4 ,4 '3 4 '-4 ' .4 4 32 4 4- .. 4 4 4 .. 4 .. 33 .. 4 .. 3 3 4 .. .. 3 3.. 4 4- .. .. 3 4 4 4 3. 35 .. 4- .. 3 3 4 .. 4 3 36 .. 4 4 4- 4- 4 4- .. .. 3A 0 0 0 2 4 0 0 0 O. :0.B 6 6 6 4 2 6

,.6c .

..6 ,;6. '6.

(b).Hibiscrub-I 4- .. 4 3 3 4 2 I I I2 4- 4 .. 4 4 4 3 2 I 13 4 4 4 4 4- 4 3 i .I i

.4 4 4 4 .. 3 4- 2 i 1 0....:5 4- 4 4 4 3 4 3 2 1 1

.6 4 4 4- 4- 4 4 3 2 2 2

"- r C 0 0 0 t 3 0 6 6 ' 6. 6.D 6 6 6 5 3 6 0 0 '-0. O.D01& 2 2 2 0 6 2 2 2 2 2Sign. Xo No No No No No Yes Yes Yes Yes

Do-&, Critical frequency value for significance at ().· Bas on a .c 3/ ~ 3 classification of the 2 x 2 contingency table.

""with the reciprocal ratings obtained with Hibiscrub.~ .by a 2 x 2 contingency table(Finne).. 1948), as follows .

Vinson's ratings,..

'd. "_~ ...4 4Aquaspt& A. BHibiscrub& CD

~r:66

12

: \1.

r -Õ.'"

-"';."'

". "'-HiíerA:B;()).and"Dare ratingfrequencies.Sincet~'l#quenciesare to small,. 'fdra,r test, the Fisher-Yates test ofsigni~cance wa~applie!:In the gloved hand'..,~~o:$tatistica.il~'.significant differençe bet~:een"thedh:g~a~p(& and HibisCrub"

'.smples \\ás detected. .-In' the unglo\'eçlhand, apar f~öir..,the ratings obtained~Jrfniediately after the antiseptic hand wash procooLlfe(t¡fft'i)), the two rcgimens~e~hibited "inson's ratings at .i, 2, 3 ,and ,4 h which,stà,tïstlcàlly, are significantlydifferent. ,k" ; , ... The "lnson's growth inhibition ratings (4-0) of S. aureus. after 3 min hand washwitb either Aquaseptl or Hibiscrub& were similarly analysed and presented in

Table 2. In theglow!d1iand, apart from the ratings obtained at time 0 and 4, at1,2 and 3 h the two regimens exhibited ratings which differed statistically sig.nificantly. In the unglo\'ed hand the ratingsdiffered significantly at each timeperiod.

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;J42 C:A.:13:ARTZOKAS AND .OTHERS\

.. Table':2..l"ins '8'fng'èr imprint rati,ig8"ot'er~4 h, Jollowing3min.halidwa8h with. Aquaept$ or H ibi8Ciub" " versus

Staphylococcus aureus '

"'Gloved hand (h) 'Fngloved hand (h)~ ,~.., .

Vol. .. ,0 ..2 3 .4 0 .2 3 4

(a) .Aquaspt-1 4, " 4 4 4 4 4 4 4 4

2 4 4 4 4, 4 " -4 .4 4 -4

3 -4 4 -4 4 . 3 4 -4 -4 4 3

-4 -4 4 -4 3 3 -4 4, -4 4 3

5 -4 4 -4 3 3: 4 " -4 4 3

6 -4 4 -4 -4 -4 4 -4 4, ,4 4

A 0 0 0 2 O. ::,.:;0:; 0 0 ZO O.

B 6 6 6 4 6. 6 0.6 6 .6 6.

(h) Hibiscrub-1 3 3 2 2 2 3 1 0 0 0

2 3 3 3 3 ' 3 3 1 0 0 0

,.3 3 3 3 .3 "3 3 1 0 0 0

4 -4 3 3 .. 3' a 3 1 0 0 0

5 -4 4 3 'S" 3 3 'I 0 0 0

....6 .3 .3.,;:3, ,:3,-4 ::.3.. I 1 0 0

','C . '.~.3' . .5:. ~ .C,' "'~ "..:",1. d .. ':-,,6 ".''6 6 6 '6.

D 3 1 0-.0 ' .'5. 0 .0 0 0 O.

DO-i 2 2 2 0 2 2 2 2 2 2

Sign. No Yes Yes Yes Xo Yes Yes Yes Yes Yes

D_. Cntical frequency vahiefor significance at 0"5.. Bas on a ~ 3/ ~ 3 clasifieation of the 2 x 2 contingency table,

,. -I

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. .. 'DISCUSSION

Since bacterial growth is promote under the occlusion of gloves and penoratedgloves unnotice during surgery (Lowbury & Lily, 1960: Walter & Kundsin.

1969; Church & Sanderson, 1980fhave ben implicated in post-oper~tive sepsis,a long lasting antiseptic activity sems a useful feature of surgical hand disin-

fection (LOwbury & Lily, 1973; Reber et al. 1975, Bartzokas el al. 1983a). The. .' .antiseptic..regim:ens. studied

in Experiment 1 appeare equally effcient whente~te. immeaiately after use: 'ii. 3

min energetic band wash reduced the normal skin

fiôra byonelôglo.Aturthèr two applicationsofthe same antiseptics, formulated'.. .inisopropyl alcohol, achievèdan almOst three logio reduction. A rè'duction of

. ~bout one log~'o :lli:th'e resident fiora,.pers to be' the norm for skin. disinfect ion

with cleansing preparations. A 2'~310gio reduction in Europe is a prerequisite for. effcient surgical disinfection with:alcohol-based rubs (Rotter, Koller & Wewalka,1981). '

Artificially a.pplied transient bacteria vary in their ability to sun'in on theskin: most Gram-negative b&cillLdie'lapidly. Ayli

ffe , Babb & Lily (1981) sug-

gested that' an organism which can ~ distinguished from t,he nonnal skin flora.such as a pigmented strain of coagulase-negative micrococcus, would

be ideal if its

resistance to skin disinfectants is similar to that of Staphylococcus aureus '. The

artificially inoculated M. roseus was completely removed from the skin. Despite

.'r:'~ f

,Bkin antiseptics:.immediateand sustained action 343two .further inocula, this markC'r was nof reco\"C'red from

'any post.tl'atment

, samplings.IThis may ,have brn due to its sust'ptibilty to,thC' antiseptics tested,

. ,'Or,'perhaps :an:altere adherence on the skin.

"In Experiment 2,f;loved'handssimulatedlh~-moist; warm environment create. ". '. ""during:surgery;,unglovedhands.the skin condition

between ,hygienic hand dis-

",.". infections. To as remanent~ffect~ S;epidermidisrepresentingnormalflora. and

methicilin-resistant 8,aureus as a menibgrofthe transienHlora, 'were used.

When

either antiseptic was challenged with S. epidemiidis, no statistically significant;-,..... .. ,."'.different remanent 'effect was demonstrate on the gloved hand over 4 h. In the

unglo\'ed hand. however, the remanent effect of Aquaspt~ remained unaltere forup to 2 h, diminishing onlyslightl)' over the.following 2 h, whereas the effect ofHibiscrub~ was progrssively

reduced from time o (Table 1). When triclosn andchlorhexidine gluconate were challenged with S;ti:ureus, their

different remanent

effects were amplified: in the unglovedh~nd thereitanent effect .of triclosan wasclearly superior to that of chlorhexidine, even immediately after

a 3 min handwash

(time 0). The difference of remanent effect between triclosan and chlorhexidine

gluconate, depending on whether gloves were worn, has not ben pre'-iously

describe~ The possible enhancement of this effect, in relation to the occluded skin,

. is being investigate. ,c.Thecrmanent..eectofchlorhexidine gluconate against reident flora has pre-

viouslyben Studied 'up to 6 h following hand disinfection (Aly & Maibach, 1979;

Werner & Borneff, 1980; La Rocca & La Rocca, 1982),but in occluded hands only;

its effect in unoccluded hands' has not ben evaluate longer than 1 h post-

application (Lowbury, Lily & Ayliffe, 1974; Peterson, Rosenberg & Alatary, 1978;Aly &Maibach~ 1980). In this 'åtudythe concentration of triclosan, deposited

on

the skin, was'suffcient to inhibit 10' c.f.u./ml of S. aureus and S. epidermidi8 after30 s contact. In a previous study' (Bartzokas et al. 1983a) another triclosan pre-paration; similar to Aquaspt~,reuce by 350fold a 2'5 x 10' forearm inoculumof an antibiotic-reistant

Klebsiella.;aerogenes in 2 h.Prolonged activityagMiist/hand-'rnediate hospital pathogens can have prac-

tical applications. Unlike chlorhexidine. (Brumfitt, Dixon & Hamilton-Miler,1985), triclosan remains active

against all S. aureus strains tested in this labora-

tory, regardless of theirsusceptibi1it~. to antibiotics. The rapid, sustainedbac-

terial attrition exhibite b)' trieloSa against importantskintrBJients can.there-fore; counteract inevitable 'lapses 'in :frquency' and

proceure ;inhandwashing by

hospital staff (Taylor, 1978r ;Thesustained,:activity of hand disinfectants,cos-metically appealing to staff aridpatients.(Slade;Wiliams,& Bartzokas,1986), canafford simple. and inexpensive 'prevention ()fhand-mediated sepsis. However

it isJ ~ _,', " ., - .' . -

their judicious application' and tbecontinuous education of clinical staff, ratherthan their remanent effectper.se, that can effectively prevent hospital infections.

t '.'

_.._-_.__..!_'

c,

REFEREXCES

.An,R.' l iiAIBACH. H. 1:(1979). Comparative study on the antimicrobial effect of 0'5%

chlorhexidint" gluconate and 70% isopropyl alcohol on tht' normal flora of hands. Applied andEn i'i ronnienlal JI icrobiology 37. 610~ 13.;' ~ i,

An. R. & iIAIBAC'H. H, 1. (1980). A comparison oftht' antimiaobial effect ofU'5 % chlorht'xidine '!(' Hibistat') and 700'0 isopropyl alcohol on hands contaminatt' with Serratia marcesceM,Clinical Experimental Dermatology 5, 197-201.

!.... .;. --;"'

:'844: ',;" ;.',,1' ...:"C.~A.' BARTZOKASAND OTHERS

, AYLIFFE,G.A J.: RUB. J. R. & LILLY; H. A. (1981), Tests for Hand Disinfection. In Dis.

.:;'iflJtctnU,:,~ir';tu~:øit tvaUaio of tfftdii.tntSS (ed. C.ll. Collns. M. C. Allwoo, S. F.

.~Bloomfield ..d~A.~Fòii);pp.,37-59. 'London: Academic-Pre..', .':llA,RTZOKAS,:.C.Â.,-CoILL,J. E.. MAKIN, 'T. ..t'PiNDER, .D.'C;''(l983ø).AlImentof the

'. 'rèir.anentantibäCrial effect ofa'2%,triclosn-detergentprepaation on the skin. Jiinul of. ;, 'JlygÙM 91, M 1.,528... . .

:BAllTZOKA8,' C,. A':,' GIBSON;:M. 'F.; GRARAJ(;~R: ':a: 'Pn~'DER.'D:'C.'(1983b). Acompari80n of- .: trido~n andchl,orhexidine preparations with 60 per cent isopropyl alcohol for hyginic hand

... Aisinre~tion. Jounul of H08IJital Infection 4, 245-255.

. ',BRUMFIT,W.. DIXON.S. &. HAMILTON-MILLER, J. M. T. (1985). Resistance to antiseptics in.'- -, methicilin.and gentamiein resistantStaphylotu øuretu. La1l.i, .1442-1443.

CHURCH. J. & SANl)BBSN, P. (1980). Surgical glove punctüres. Joimia of H08pitalInfection1,'84.. ... .FBDBRAL REOISTER (1974). OTC Topical antimicrobialproucts.j'tdral Regi8ler 39(179),33140.'. ...... . .t;;; .FEDERAL iRzoisTBR (1978). OTe Topical AnÍímÎërbial Prou~. Fedral Regstr 43(4),1215. . ¡'.:..',.'..., . .,.,..".,~".,;~FINNBv, D.J.(i~). The Fisher-Yate tet of sigrîßcince in2',f2oontingency tables. Bio-.. ~ril: 35, 145156. .LA RoA.,'M.' A. & LA RoA, P. T. (1982). An evaluation of the antimicrobial effect ofa hand

8pnge~brsh'rimpregnate ,with '% chlorhexidine.gluconate (Hibiclens). Detl()ments in"'Uflturu'.Miuy2J, 54~M6. . . . .' ". .LowBuav, E. J~L. & LiLV. H. A. ~196). DisÎnféctjòlH)f thehand8"Of 8Urgeons andnur8s.Briiøh Medlc JourTl. 1445-1450. '. .

'LOwBÚBt.:~IE/J:'L.-'&.'LILy,H;"A.'- (1973);"': Use- of;.%.',chlorhexicin~ detergnt solution.'o'(Hibisb)&nd,other method:ofskin disinfecion. Briiøh .Medic ;'ouma I. 510-515.LoWBURV, E. JiI... LILLV, H. A. &. AVL1l'B;G.A. J. (1974).Preperative disinfection of

, surgeons' hands: use of alcoholic solutions and effects of gloves on skin flora. British MedicalJoÚi7 .t: 370-372. . ':', 0 .PETidisôlt; A. F.; RoSBNBERO, A. &. AiTARv, S. D. (1978). Comparative evaluation of surgical-:scrib preparation.8urgtr. (J and ,Ob8téiri 146,63-6. .'; .

RBBBR,"R"MONTBNBR, M., NBCK, K. & LI, U, (1~75). Zur prufmethodik der chirurgeschen! ihanded~lJfektion. Zenrila fir Ba ufl HygM im ~btilu71 Orinae 160.:"-6Òì-:27'.~'." '. . ."; ;'.' -, ; " .

iRoù,'M.,iKoLLEi. W.'&. WEwALKA,G. (1981);. Eignung von chloi:hexidinglukoniit-undPVP.Jod.haltignpräparationen zur händedesinfektion. Hygie ú':'Medizin 6.425-30.

..,. ßIl;~;R-:tlQPP)"!N f!parameri S.tai8tic for.k .&M'Vi Scien.New' York: McGraw- Hilr¡;~~.~aipay: "" .-. .- '. ... .;, . ;,'SI.~B"l:D.;'WILLIAMS. E. & BARTZOKAS, C. A. (1986). Psychologci.lcontributions to the

';' :ÒÒntÍ'L~f hòëpitàlinfection. In Curren luu in Clinic P,ycA (ed. N. Eisenberg and"j;i(iD/G1Úgow),'London :.' Gower. "~ ,:&~ir,LJ.Jt~~l.;A .evaluation of- had-wasing teniques. 'Nur8Ì71 Timu 74, M-55.

. ._ ',"~qS¿UO~._....,'.. ". " . : ,"-0 ...viNSoN;L':"j~,"'ÂJiB"'YB:"E.ì.:. BENNET. A. G., ScNEIDER, W.'C.' & TRAVERS. J. J. (1961).

,."....ln~lKro~~for.mea8uring antibacterial activity of toilet 8Oap¡a.nd detergent bars. Jounul':'¡-;'~oíip1arnic Scief 50.827-:830. . .. : ". ''4Y"'i.ji..i9.;~,A.E,,-NDSIN;.R~ B. (1969). The bacteriologic study óf Ilurgical glo\'es from 250

\ìitt~~"i~~~::trr~;,~ il:8~~;t~~~i~~:5;~r'deSin~~i~renden ,,'irksamk~it"V'()~.j'h¡ìidedesinfelctionsvenahren am beispiel \"on 0'5% chlorh~xidi"ngluconat in 70% iso-., propylalkohol. Hygiene und Medizin S. 61-70.

c

(

.i~

A comparison of triclosan and cWorhexidinepreparations with 60 per cent isopropyl alcohol

for hygienic hand disinfection

( .

(C. A. BARTZOKAS, M. F. GIBSON, R. GRAHAM AND D. C. PINDER. ¡ .

(

(

(

Reprinted from the

Journal of Hospital Infection

Vol. 4, No.3, pp, 245-255

September 1983

I

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i

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u

u

Joiirnal of Hospital Infection (1983) 4, 245-255

A comparison of triclosan and chlorhexidinepreparations with 60 per cent isopropyl alcohol for

hygienic hand disinfection

C. A. Bartzokas,* M. F. Gibson,* R. Graham* and D. C.Pindert

*Department of Medical Microbiology, Duncan Building, Royal LiverpoolHospital, Prescot Street, Liverpool L7 8XW and tMedical Department,

Mersey Regional Health Authority; Wilberforce House, The Strand,Liverpool L2 7RW

Summary: Four detergent preparations containing triclosan; two alcoholicproduc:ts, containing triclosan and chlorhexidine respectively, and a detergentpreparation containing chlorhexidine were compared with 60 per centisopropyl alcohol following a single hand wash using Escherichia coli as the testorganism. In vitro tests indicated that all had a high activity against hospitalpathogens. The triclosan-containing preparations exhibited a significantresidual activity. Experiments in volunteers showed that 0'5 per cent alcoholictricwsan was significantly more effective than all other preparations tested. Allthe detergent preparations had an effect similar to that of 60 per cent isopropylalcohoL. None of the seven products evaluated was significatly less bacteri-cidal than 60 per cent isopropyl alcohoL.

Introduction

In the last 30 years the detergent and cosmetics industry has been adding, atone time or another, hexachlorophane, tribromosalicylanilides, halogenatedcarbanilides, quaternary amonium compounds and chlorhexidine gluco-nate as antibacterials to soaps, detergents and deodorants, but relatively fewhave been accepted for hospital usage.

Hexachlorophane has been extensively used in medicinal topical prepara-tions but the ecological influence of this compound on the skin microflora hasbeen criticized (Evans et ai., 1973) and the possibility of toxic absorption hasled to regulation of usage (Drake, 1974; Hopkins, 1979).

Chlorhexidine has gained acceptance as an alternative (Smylie, Logie andSmith, 1973) but its effcacy in hand disinfection has been questioned whencompared with 60 per cent isopropyl alcohol (Rotter, Koller and Wewalka,1980). Furthermore, a contamnated chlorhexidine hand wash solution hasbeen associated with an outbreak of nosocomial infection (Marrie and

Costerton, 1981) and species differences in resistance to chlorhexidine havebeen described (Nakahara and Kozukue, 1982).

Another antimicrobial agent now gaining favour is triclosan (Furia and

0195-701/83/04245 + II 502.00/0 iO i 983 The Hosp;..1 Infecon &i¡ety

245

246 C. A. Barlzokas et aI.

Schenkel, 1968) probably acting on the microbial cytoplasmic membrane(Regös and Hitz, 1974). Unlike hexachlorophane, triclosan is active againstthe enterobacteria, a wide range of Gram-negative intestinal micro-organismsand skin flora, including Staphylococcus aureus (Vischer and Regös, 1974).With no reported adverse .toxicological constraints, triclosan has replacedhexachlorophane ~n most toiletry and cosmetic preparations. In comparisonwith cationic chlorhexidine salts, triclosan offers more formulation flexibilityand can be used in anionic tenside systems, such as soap, without apparentloss of effcacy.

Triclosan (2,4,4-trichloro-2-hydroxydiphenyl ether) preparations have

been evaluated for surgical hand washing (Lilly and Lowbury, 1974; Eitzenet al., 1979; Cremieux, Guiraud-Dauriac and Dumenil, 1980), whole bodybathing of acute leukaemics kept in protective isolation (Bodey and Rosen-baum, 1973; Bodey, Ebersole and Chung Hong, 1976), as bath additives forthe treatment of staphylococcal carriers and in combination with cortico-steroids in secondary infected eczema and epidermomycosis (Reiffers, 1981).

We report here a study in which triclosan and chlorhexidine preparationswere compared with a control, 60 per cent isopropyl alcohol, in vitro and involunteers, for effectiveness in hygienic hand disinfection. Seven com-mercially available products were evaluated as follows: five triclosanpreparations (four in detergent and one in alcohol) and two chlorhexidine(one in detergent and the other in alcohol).

Materials and methods

Test formulations1. 'B.P: containing 0.5 per cent triclosan in detergent (supplied by B.P.

Detergents Ltd),2. 'Florafree' containing 0.3 per cent triclosan in detergent (supplied by

D.E.B. Chemical Proprietaries Ltd),3. 'Sumasept containing 0.5 per cent triclosan in detergent (supplied by

Lever Industrial Ltd),4. 'Zalclense' containing 2 per cent triclosan in detergent (supplied by

Sterling Industrial),5. 'Manusept containing 0.5 per cent tric10san in 70 per cent isopropyl

alcohol with added emollents (supplied by Hough, Hoseason and Co. Ltd),6. 'Hibisol' containing 0.5 per cent chlorhexidine gluconate in 70 per cent

isopropyl alcohol (supplied by ICI Pharmaceuticals pic), ,~-7. 'Hibiscrub' containing 4 per cent chlorhexidine gluconate in non ionic

detergent base (supplied by ICI Pharmaceuticals pIc).

In vitro testsThe following pure cultures were used:

Staph. aureus, methicilin-sensitive (NCTC 4163),Staph. aureus, methicilin-resistant (R.L.H. clinical isolate),

Hand disinfectants 247

Streptococcus faecalis (NCTC 8619),Escherichia coli (NCTC 9001),Klebsiella aerogenes, gentamicin-resistant (R.L.H. clinical isolate),Pseudomonas aeruginosa (NCTC 8060),Proteus vulgaris (NCTC 4175),Bacillus subtilis (NCTC 8236).

The seven test formulations, controlled by 60 per cent isopropyl alcohol,were tested against selected bacterial pathogens for antibacterial activity:residual, using calf-skin as a model substrate and intrinsic, using an agar-cup diffusion test. Twenty ml of nutrient broth No. 2 (Oxoid CM 67)containing 105-106 cfu/ml of each strain were inoculated on to assay plates(23 x 23 cm) containing Columbia Agar (Oxoid CM 331).

Calf-skin halo test (Vinson et al., 1961). A 2 cm diameter disc ofdehydrated sterile calf-skin was immersed for 1 min in a 50 per cent solutionof each test formulation in sterile Liverpool tap water, rinsed under runningcold tap water for 1 min, blotted dry and placed on the previously inoculatedassay plate. Following incubation at 3JOC for 24 h the zones of growthinhibition (halos) in mm around the discs were recorded and any growthunder the calf-skin disc was noted.

Agar cup test. Holes, til in diameter, were cut, in the agar on the assay plate,with a corK borer and the cavity sealed with a few drops ofmo1ten agar: 0.2 mlaliquots of a 50 per cent solution of each test formulation, prepared as above,were placed in duplicate. Following incubation at 37°C for 24 h the diameterof the zones of growth inhibition in mm were recorded.

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Hygienic hand disinfectionMaterialsInoculum. A 5 ml aliquot of a 24 h culture of E. coli (ATCC 11229) grownin nutrient broth No.2 (Oxoid CM 67), inoculated into a further 2 i of thesame broth and incubated for a further 18-24 hat 37°C to obtainapproximately 108 cfu/mL.

Kneading fluid. 1 per cent peptone water (Oxoid CM 9).Deactivator solution. 1 per cent peptone water, 3 per cent Tween 80, 0.04per cent sodium thioglycolate.Recovery medium. Columbia agar (Oxoid CM 331).

Methods (based on that of Rotter et al., 1977; Ayliffe, Babb and Lily, f981)Each of the seven test formulations was assessed separately on fivevolunteers. All volunteers had short to normal length finger nails, no visibleskin injuries, eczema or other skin disease. During testing no rings or wristwatches were worn. Rinsing was under lukewarm Liverpool tap water(bacterial count -c 1 cfu/ml) and, except following the artificial contami-nation, hands were dried with sterile paper towels.

1. Initial hand wash. All volunteers washed their hands with a non-

248 C. A. Bartzokas et aI.

medicated, preservative-free liquid semi-synthetic detergent for 2 min,

rinsed for 20 sec and dried.2. Artifcial contamination. Both hands were simultaneously immersed up

to the middle of the palm in 2 I inoculum placed in a previously sterile 6 ibasin. After 2 min immersion, the hands were withdrawn and air-dried for3 min; both hands were rotated arid the fingers flexed continuously to avoiddroplet formation on the finger tips. .

3. Pre-value determination (V w). Each hand was placed simultaneouslyover a Petri dish containing 10 ml of kneading fluid, and the fingers werekneaded continuously for 1 min. Immediately after, the kneading fluidsfrom the left and right hands were serially diluted in deactivator and 0.1 mlaliquots of neat and 1 :10, 1: 100, 1 :1000, 1 :10,000 and 1: 100,000 dilutionswere spread over the surface of Petri dishes containing recovery medium. Allplates were incubated at 37°C for 48 h and the efu in plates containing30-300 cfu were enumerated. Deactivators were not included in the knead-ing fluid for V w determination, since deactivator residues on the fingers couldpotentially interfere with the effcacy of the antiseptics applied subsequently.

4. Post-value determination (N J~a) Standard antiseptic evaluation. Immediately after the kneading of the

fingers for the pre-value determination and without drying, an aliquot of3 ml 60 per cent V IV isopropyl alcohol was applied to cupped hands at 30 sintervals while volunteers rubbed their hands to dryness for 1 min followinga standard procedure. The hands were then rinsed for 20 s and dried. The N wwas determined as for V W' except that the kneading fluid containeddeactivator and was diluted up to 1: 100 only, thus plating 0.1 ml aliquots ofneat, 1: 10 and 1: 100 dilutions.

(b) Test antiseptic evaluation. Test antiseptics were applied under thesame standard conditions rather than specifically to manufacturers in-structions in order to compare their relative effcacies more readily. Within2 h from the first arficial contamination, both hands were contaminatedagain and the V w determined as above. Immediately after the v w de-termination, the hands were wetted under running water and two aliquots,each of 3 ml of the test antiseptic, were applied at 30 s iIitervals andvolunteers rubbed their hands to dryess for 1 min, rinsed for 20 s and dried.The Nw was determined as described under 4(a).

Results ,.;

1. Calf-skin halo testA persistent or long-lasting antibacterial action on the skin between hygienicwash treatments may be of value in reducing the spread of transient hospitalpathogens. The claimed skin substantivity of triclosan and chlorhexidinemay be investigated in vitro, using calf-skin as a model substrate (Vinson etal., 1961). Active antibacterials persisting on calf-skin following thewash-rinse treatments, inhibit the growth of bacteria around andlor under

Table i. Calf-skin halo test

Org

anis

m (

zone

s of

gro

wth

inhi

bitio

n in

mm

)

Staph. au

reus Staph. au

reus

K. aerogenes

met

hici

lin-

met

hici

lin-

gent

amic

in-

Prod

uct

sens

itive

resi

stan

tSt

r. f

aeca

lisE

. col

ire

sist

ant

Ps.

aer

ugin

osa

Pro

vulg

aris

'B.P

.'53

380

2427

040

'Flo

rafr

ee'

4436

024

240

38'S

umas

ept

5141

032

290

42

'Zal

c1en

se'

6145

2236

3-3

047

60%

Iso

prop

yl a

lcoh

ol24

240

00

00

'Man

usep

t50

510

3731

047

'Hib

isol

3031

2727

2323

22

'Hib

iscr

ub'

3831

2727

2425

25

Bla

nk s

oap

(no

antim

icro

bial

)0

00

00

00

.',

.._-.

._--

--..~

.....~

..~~~

..._'

~..-

-_..-

_.,-

--.-

,.

Tab

le I

I. A

gar

cup

test

Org

anis

m (

mea

n of

the

zone

s of

gro

wth

inhi

bitio

n in

mm

)

Staph. aureus Staph. aureus

K. aerogenes

met

hici

lln_

met

hici

lin_

gent

amic

in_

Prod

uct

sens

itive

resi

stan

tSt

r. ¡

aeca

/isE

. col

ire

sist

ant

Ps. aeruginosa

Pro

vulg

aris

'B.P

.'52

4721

35'5

38.5

19.5

44'5

'Flo

rafr

ee'

44,5

41'5

34'5

27'5

3016

'540

.5'S

umas

ept

57;:6

034

3134

,519

45'Z

alcl

ense

'63

;:65

36.5

3641

'518

57,5

60%

Iso

prop

yl a

lcoh

ol39

'545

'50

21.5

26'5

17'5

40.5

'Man

usep

t;:6

572

'525

'542

40'5

19'5

61'H

ibis

ol36

3836

35.5

3048

45,5

'Hib

iscr

ub'

3231

'532

2529

3433

Bla

nk s

oap

(no

antim

icro

bial

)0

00

00

00

- ~


, the skin discs. The diameter in mm of the zones of growth'inhibition (halos)around the calf-skin discs are presented in Table i. Where no inhibitionzones were evident, no growth under the calf-skin discs was detected.

2. Agar cup testThe agar cup technique was used to indicate the intrinsic antibacterial actionof triclosan- and chlorhexidine-containing preparations against a range ofhospital transients, including antibiotic-resistant pathogens. The mean ofthe zones of growth inhibition in mm are presented in Table II.

3. Hygienic hand disinfection .The total bacterial counts for the left and right hands of each volunteer wereaveraged, transformed to logio, and thelogio (mean Vw)-logio (mean Nw)was calculated for both control (60 per cent isopropyl alcohol) and each of thetest antiseptics (proprietary products). The logio reductions in skin bacterialcounts obtained for each of the five volunteers and for each of the seven pairsof standard alcohol-proprietary products tested, are presented in Table III.This shows that only four of the seven products ('Sumasept', 'Zalclense','Manusept' and 'Hibisol) produced the required thousand-fold reduction inbacterial counts on the skin of 80 per cent of volunteers, specified by Rotter etaL. (1977).

4. Statistical analysis (Rotter et al., 1977)

The difference in (logio) bacterial reduction between the products andalcohol for each of the seven products and five volunteers are given in Tableiv, together with the row and column totals. This shows that of the abovefour apparently satisfactory products, only two ('Manusept' and 'Ziilclense')performed as well as alcohoL.

An analysis of variance was performed to determine whether there was astatistically significant difference among the products. The stages are set outin Table V. This shows that there was no significant difference among thevolunteers, but that there was a highly significant difference among theproprietary products (F(6'24)=6'36, PooO'l per cent).

In order to determine whether any product was significantly better thanany other, allowance must be made for the fact that 21 different pairs can bedrawn from the seven products. In comparing any two mean product-alcohol ,~differences (obtained from the row totals of Table IV) the difference betweenthem must therefore be greater than the usual two standard errors. A pooledvariance, and hence standard error, for the product means is calculated fromthe data in Table V:

Standard error for mean product-alcohol difference = 4.6801 +0.8353(24+4)5

=0.1985.

252 C. A. Bartzokas et al.

Table II i. Log i 0 reductions In bacterial counts: logio (mean V J -logio (mean NJ =Reduction Factor (RF)

VolunteerStandard alcohol-product comparison i" 3 4 5

Alcohol 4.0607 2.4210 3.0207 2.8366 3.3090'B.P.' 2'9700 2.4796 3.1450 3.0747 3.1389Alcohol 3.9201 3-670 3'1486 3.6065 2'5014'Florafree' 3.0.148 3.0542 2'5333 3.3322 2.7482Alcohol 3'4117 3.4680 2-128 3'1858 3'3194'Sumasept 3.0725 3.2865 2'3458 3.0094 3'5782Alcohol 3.7868 2.8706 3.1448 3.0769 4.2272'Zalclense' 4.0911 3.8812 3.0799 3.2882 .3-630Alcohol 2'5589 3-663 2.2683 2-613 3.0669'Manusept 3.5938 4.2788 3.5986 3.7939 4'8016Alcohol 3.4084 3'5464 4.2484 2.9559 3.6902'Hibisol 3'5240 3.5880 4.3926 2.9667 3'1584Alcohol 3'4017 2.1519 3.4046 2'7986 2.9836'Hibiscrub' 2.8797 3'0067 3.3550 2.7007 .3.2175

Table iV. Diferences in logio reductions in bacterial counts between products and alcohol

Volunteer

Product 2 3 4 5 Row totals

'B.P.' -1.0907 0.0586 0'1243 -0'2381 -0'1701 -0,8398'Florafree' -0.9053 -0'6128 -0.6153 -0.2743 -0.2468 -2'1609'Sumasept -0'3392 -0.1815 -0'1670 -0.1764 0'2588 -0.6053'Zalclense' 0.3043 1.0106 -0.0649 0.2113 -0'5642 0.8971'Manusept 1.0349 0.6125 1'3303 0.9326 1.7347 5.6450'Hibisol 0'1156 0.0416 0.1442 0.0108 -0,5318 -0'2196'Hibiscrub' -0'5220 0.8548 -0,0496 -0.0979 0'2339 . O.U92Column totals -1.4024 1'7838 0'7020 0.8442 1.2081 3.1357

Table V. Analysis of van'ance

Source of Corrected sum Degrees of Mean .;variation of squares freedom square F-value.

Product 7.442 6 1'2410 6.36Volunteers 0.8353 4 0.2088 1.07P~sidual 4.6801 24 0.1950Total 12.9617 34


For seven products and 28 degrees of freedom the critical values for a two-tailed test are: 5 per cent, 4.49 standard errors; 1 per cent, 5 ,45 standarderrors. Important comparisons are those between the best and second-best,and between the second-best and worst products. Thus:

'Manusept-'Zalclense' = 0.9496 = 4.78 standard errors'Zalclense'-'Florafree' =0,6116 = 3.08 standard errors.

The difference between 'Manusept and 'Zalclense' (or any other product) istherefore significant (P,5 per cent), whereas the differences between anytwo products (not .including 'Manusept) are not significant.

Discussion

When the hand disinfectants were applied to calf-skin under simulated handwash conditions to ascertain residual antibacterial effect on the human skin,all triclosan-containing products were equally effective against bothantibiotic-resistant and -sensitive test strains. Str.faecalis and Ps. aeruginosawere not, however, inhibited by alltriclosan-containing products.'Hibiscrub' and 'Hibisol' shòwed a similar anti-bacterial activity against allthe strains tested. Isopropyl alcohol had a limited residual activity againstStaph. aureu. Overall, 'Zalclense' and 'Manusept were the most inhibitory.

In the Agar Cup test, all triclosan-containing products were inhibitory toall the strains tested. Generally, the size of the zone of inhibition can berelated to the concentration of triclosan in the product. Both of thechlorhexidine-based prodùcts were also active under these test conditions.Surprisingly, 60 per cent isopropyl alcohol itself, inhibited all the strainstested except Str. faecalis.

On the hands contaminated with E. coli, all seven products tested showedsignificant disinfecting action, similar to that of 60 per cent isopropyl alcohoL.The statistical analysis of the bacterial counts indicated that 'Manuseptperformedsignificant1y better than the other six (mean difference from thestandard 1'1290). In terms of rankig, 'Zalclense'was the second best

although the difference between 'Zalclense' and 'Florafree', for example, wasnot significant (mean difference from the standard -0,4321).

The methodology adopted in our experiments to evaluate the handdecontaminating effcacy was based on that of Rotter and Ayliffe (Rotter etal., 1977; Ayliffe, Babb and Lily, 1981). The former has now been adoptedin principle by the DGHM in West Germany (Bomeff et al., 1981). Similarprotocols have been advocated by the Food & Drug Administration in theUSA, using Serratia marcescen or B. sutilis var. niger as marker micro-organisms (Federal Register, 1978). E. coli was preferred in our experimentsas it is a common skin transient of faecal origin and a reliable indicator of lowstandards of personal hygiene. In addition, like Ser. marcescens, E. coli can bereadily monitored under experimental conditions using selective solid

,,iI

I

i

ì

1

I

Iii

254 C. A. Bartzokas et al.

media. However, the use of additional test organisms may be desirable(Ayliffe, Babb and Quoraishi, 1978), while applied transients may vary intheir ability to survive on the skin, equally impårtant they may also differregarding ease of mechanical removal (A. R. Cox, personal communication):following a 2 min hand wash with water alone, E. coli was more readilyremoved than either Ps. aeruginosa or a streptomycin-resistant Staph. albus.The three strains tested also differed in their susceptibility to alcoholdisinfection:

Treatment logio reduction factor

E. coliWater alone60% Isopropyl alcohol

2.573.84

Ps. aeruginosa

2'084'40

Staph. albus

2.024.34

The logio reductions obtained in our experiments should only be consideredin terms of relative effcacy compared with a standard, such as isopropylalcohol, and should not be regarded as absolute values for decontaminationactivity: since it has been shown that variations in the test protocol mayinfluence results (Hall, 1980). Furthermore, comparisons of hand disinfec-tants should be limited to observations of overall product effcacy rather thanby being related to the concentration of the bactericidal agent incorporated.

In conclusion, of the alcohol-based hand disinfectants studied O' 5 per centtriclosan in 70 per cent isopropyl alcohol ('Manusept) was significantly moreactive than both the standard 60 per cent isopropyl alcohol itself and O' 5 percent chlorhexidine in 70 per cent isopropyl alcohol ('Hibisol). The formerwould thus be appropriate for rapid hand disinfection before asepticprocedures. The benefit of this higher degree of activity should be furtherevaluated under practical conditions of use. Of the detergent-based disinfec-tants containing either triclosan or chlorhexidine, choice is probably a matterof personal preference, since all five performed similarly to the standardalcohol disinfectant. Additional characteristics of these products in relationto skin deposition following hand washing are currently being investigated.

ReferencesAyliffe, G. A. J., Babb, J. R. & Quoraishi, A. H. (1978). A test for 'hygienic' hand disinfection.

Journal of Clinical Pathology 31, 923-928.,Ayliffe, G. A. J., Babb, J. R. & Lily, H. A. (1981). Tests for hand disinfection. In

Disinfectants: TheiT use an evaluation of efectiveness. SAB Technical Series No. 16,37-4. Academic Press.

Bodey, G. P., Ebersole, R. & Chung Hong, H.-S. (1976). Randomized trial of a hexachlor-ophene preparation and P.300 bacteriostatic soaps. Journal of Investigative Deratology67,532-537.

Bodey. G. P. & Rosenbaum, B. (1973). Evaluation of a bacteriostatic soap, P-300, on skin floraof patients in protected environments. CurTent Therapeutic Research 15, 253-260.


Borneff, J., Eggers, H.-J., Grun, L., Gundermann, K.-O., Kuwert, E., Lammers, Th.,Primavesi, C. A., Rotter, M., Schmidt-Lorenz, W., Schubert, R., Sonntag, H.-G.,Spicher, G., Teuber, M., Thofern, E., Weinhold, E. & Werner, H.-P. (1981). Richtlinienfür die Prüfung und Bewertung chemischer Desinfektionsverfahren. Zentralblatt fiirBakteriologie und Hygiene I, Abteilung Originale B 172, 534-562.

Cremieux, A., Guiraud-Dauriac, H. & Dumenil, G. (1980). Activité dune mousseantiseptique sur la flore normale des mains. Médecíne et Maladies Infectieuses 10, 4-7.

Drake, J. J .-P. (1974). Hexachlorophene. Food and Cosmetics Toxicology 12, 563-568.Eitzen, H. E., Ritter, M. A., French, M. L. V. & Gioe, T. J. (1979). A mic:robiological in-use

comparison of surgical hand-washing agents. The Journal of Bone andJoint Surgery ( Am)

61A, 403-406.Evans, Z. A., Rendtorf, R. C., Robinson, H. & Rosenberg, W. (1973). Ecological influence of

hexachlorophene on skin bacteria. Journal of Investigative Dermatology 60, 207-214.Federal Register (1978). Test for health care personnel hand-wash effectiveness. Federal

Register 43, 1244.

Furia, T. E. & Schenkel, A. G. (1968). 2,4,4'-Trichloro-2'-hydroxydiphenyl ether. New,broad spectrum bacteriostat. Soap and Chemical Specialities 44,3-17.

Hall, R. (1980). Povidone-iodine and chlorhexidine gluconate containing detergents fordisinfection of hands. Journal of Hospital Infection 1, 367-368.

Hopkins, J. (1979). Hexachlorophene: more bad news than good. Food and CosmeticsToxicology 17, 410-12.

Lily, H. A. & Lowbury, E. J. L. (1974). Disinfection on the skin with detergent preparationsof Irgasan DP 300 and other antiseptics. British Medical Journal 4, 372-374.

Marrie, T. J. & Costerton, J. W. (1981). Prolonged survival of Serratia marcescens inchlorhexidine. Applied and Environmental Microbiology 42, 1093-1102.

Nakahara, H. & Kozukue, H. (1982). Isolation of chlorhexidine-resistant Pseudomonasaeruginosa from clinical lesions. Journal of Clinical Microbiology 15, 166-168.

Regös, J. & Hitz, H. R. (1974). Investigations on the mode of action of Triclosan, a broadspectrum antimicrobial agent. Zentralblatt für Bakteriologie und Hygiene, 1, Abteilung

Originale A 226, 390-01.Reiffers, J. (1981). Essai dune nouvelle crème antifongique et antibacterienne à base de

triclosan (CGP 433-Logamel 1 ~). Schwizer Rundschau Medizinische (PRAXIS) 70,1050-1053.

Rotter, M., Koller, W., Kundi, M., Wewalka, G. & Mittermayer, H. (1977). Testmethode fürdie Wertbemessung von Verfahren für die hygienische Händedesinfektion. 1. Teil:Beschreibung der methode. Zentralblatt für Bakteriologie und Hygiene 1, AbteilungOriginale B 164, 498-506. .

Rotter, M., Koller, W. & Wewalka, G. (1980). Povidone-iodine and chlorhexidinegluconate-containing detergents for disinfection of hands. Joumal of Hospital Infection 1,149-158.

Smylie, H. G., Logie, J. R. C.& Smith, G. (1973). From Phisohex to Hibiscrub. BritishMedical Journal 4, 586-589.

Vinson, L. J., Ambye, E. L., Bennett, A. G., Schneider, W. C. & Travers, J. J. (1961). InTJtrotests for measuring antibacterial activity of toilet soap and detergent bars. Journal ofPharmaceutical Sciences 50, 827-830.

Vi scher, W. A. & Regös, J. (1974). Antimicrobial spectrum of Triclosan, a broad-spectrumantimicrobial agent for topical application. Zentralblattfür Bakterìologie und Hygiene, I,

Abteilung Originale A 226, 376-389.

\

I

I

ii 1S on th: nands, but door adequate disinfection..udies at least 3 -5 ml of'lfécllOn_ The practice ofnfect the hands duringsinfection of the hands.alcohol can be used forhe hands \\ Ith soap and

A test procedure for evaluating surgical handdisinfection

J. R. Babb, J. G. Davies and G. A. J, Ayliffe(

-Hospiial Infeciion Research LoboratMY, Dudley Road Hospital, BirminghamBI8 ìQH, CK

t\. of soap as '.ariable in

Summary: .\ technique for assessing the i",mediate and prolonged effacyof surglèal scrubs Jnd akoholic hand rubs IS de-;ribed. ..\ mean b~linecount is obtained from all \ olunteers and logarithmic reductions .n re'skin Rora immediatek after one or more appli,ations, and after ",I. Iglo'-es for 3 h, are measured Loose.titting surgical glo'.es are us.sampling resident Aora. Preparations were applied using a standardnique for 2 min, apart from one test with 70',. isopropanol (IP.-\) in ",hI'appliçation time was 30 s Two studies are described, one of which comparedfour chlorhexidine scrubs, and the seèond 70','. LP,.\. 7 5'1. pondone-iodinescrub. 2". triclosan cleanser and unmedicated bar soap. In spite of theirèonstituent slmilJrtt\', the four èhlorheXldlne scrubs ,.aried considerabh' ineffcac~ and user acc~ptabillt\.. _.\ 2 min appllèation of 70'" I P.-\ was the ~OSt

effective treatment, and gave log., redu.:tions of I 6; for immediate and \.;8for prolonged e!fcct.Thls "as marginallv more effectl\e than a 30 s applica-tion, but the difference was not signiticanl. .Hibisuub' was the most effective

aqueous formulation and gave redu.:tlons oi 1.01 for immediate effect ¡od1.16 for prolonged e!fecl. The test desuibed .:ould be used by reference

,'entres and manufat:urers to assess the eHicac\. of new and existing surgicalhand disinfet:ion formulations.

disinfection \\Ith frequent'i. 79; 107-112

ethods in remo\ ing transient

, and hospital em, roninental

j h'-giene in hospital J Hyg

(of hands with emulsion --3;9; n 3ì7-J86dryness and eczema of the

ng '\Ith soap. J. H'Jsp Infut

K(\u.o,ds: Surgical scrub, antiseptics; test methods.

Introduction

(

Considerable pressure is exerted on microbiologists and phannacists to

substantiate disinfectant manufacturers' claims and, where possible, toreduce or minimize infection risks without incurring additional expense.

Because the incidt:nce of postoperative wound infection in clean ..ounds isso low, it would prO\'t extremely diffcult to measure the effectiveness ofhandwash formulations by monitoring their influence on the incidence ofpostoperative infection.

There is no general agreement as to which of the many test methods

a\ailable is most suitable for assessing the effciency of hand disinfectionproducts.I-) Surgical scrubs are usually im'estigated by measuringreductions in the resident skin !lora. l; and ward or hygienic handdisinfection products by measuring reductions in transient microorganismsartificially applied to the hands.b,; Emphasis is currently placed on adoptingone or more of these tests as an 'i nternational Standard' ..hich would enable

( I

( iiiq~ ~:OL QII)t9i'-I.I)QJoJI1I10 ( laii ~ H~piaJ In(f'IOO SoW"

41

l

(. I

(.

.2 J. R. Ilbb et &1.

--\

I

i

I

manufacturers, purchasing authorities and reference laboratories tocompare, under realistic conditions. new with existing formulations. Twosuch tests, i.e. for hygienic and. surgical hand disinfection, have recently

been described8Q and these ..ere found to be reasonably repeatable and

reproducible in three. and four centres, respectively.The surgical test,4 which In\.ol..es the use of a glove juice sampling

method,lUll has been used here to evaluate several aqueous and alcoholichand formulations and their mode of application.

Method and materials

The test was an open. randomized. comparati\.e cross-over evaluation ofproducts, and was performed on healthy volunteers aged between 18 and

65. \'olunteers had short well-kept finger nails and healthy undamaged skin,and had a\oided using antimicrobial substances on the hands for I week

bdore and during the study. Persons receiving antibiotics, or those withknown sensiti\.ity to the products under test or having a history of skinallergy, were excluded. Lnmedicated bar soap was provided for other skincleansing during the trial period and gloves were used by the volunteers ifcontamination with other antimicrobial agents, e.g. disinfectants, solvents.acids or bases was likely.

Sampling methods and 5tudy design (Fig. I)Baseline counts. Baseline counts were obtained by sampling the hands on

three occasions with at least 48 h between each sample. The mean of theselog counts was used to determine the baseline count for each hand.

Hands and forearms were washed with unmedicated soap for 30 s, thenails cleaned with a manicure stick under running water, and the hands

rinsed well and dried with paper towels. The hands were then sampledseparately by donning a pair of loose-fitting, unpowdered, sterile surgicalgloves (Regent Biogel) and adding 50 ml of recovery broth. This consistedof tryptone soya broth with appropriate neutralizers to the product undertest, i.e. O' i 5 % lecithin-Tween mi.xture (5 g lecithin and 50 g Tween 80) forchlorhexidine-containing formulations, 1 % sodium thiosulphate forpo\'idone-iodine formulations and 3% Tween 80 and i % lecithin fortriclosan-containing formulations. The glove was sealed at the wrist withan adjustable strap. Four areas of the hands, i.e. fingers, finger webs, backsand fronts of hands were massaged using a standard technique by applyingfirm but e\'en pressure. The hand was then clenched and opened twice, andthe whole procedure repeated twice more during the 1 min sampling period.The strap was then released and an aliquot of the sampling fluid removedfor counting. The other hand was sampled in the same manner, after which

the glo\'es were removed and hands rinsed and dried.Tenfold dilutions of the sampling fluid were made in 1/4 strength Ringers

solution and 5 x 0.02 ml drops (0,1 m1) of each dilution, and an undiluted

sample. were transferred to the surface of well-dried tryptone soya agar

'terence laboratories to¡sting formulations. '1"0sinfection, have recently

asonabl\ repeatable and

Iv.a glo\e Juice sampling

al aqueous and alcoholic

:rOSS-O\.er e\.aluation of

rs aged between 18 andieal thy undamaged skin,n the hands for I week

it¡biotics, or those withaving a history of skin

pro\ided for other skin,ed by the \olunteers ifdisinfectants, soh.ents,

sampling the hands on

pie. The mean of theset fòr each hand.ated so.ip for 30 s. the

water, and the hands

ds were then sampledidered. sterile surgicalbroth. This consisted

; to the product underind 50 g Tween 80) forelm thiosulphate forand 10.0 lecithin foraled at the wrist \\ ith:rs, finger webs. backs:echnique by applyingind opened twice, andmin sampling period.

mpling Auidremovede manner, after whtih

1 I. -l strength Ringerson, and an undilutedd tr~ ptone soya agar

;~

~,. ~" c..;= '" -

i~. f.: ,. ,.~ ;.c,., ~ ~"" -= -!! : ~;: -..-II "= ë '.. ~:: ::"" ::

~ ~;1

Evaluation o( iurcical hiud disinfection

i:..'";; c:~ '- .. =.2c"" ~ ~ 2

. 5 c _ ~ .- i ~ Coc::.:i:. cc;"" c.:~~ .. .. ~ ~ ~ ='- = cf-C:;;"¡ È=E ~x~.."c.:~"I~ .\l5: 2-=ê~;~ ~ ~ E ~ - j ~ ': -1: ; g ;..~=-E:: ~..:: ~U',.":~-E--= '.;.- e.E::_e:G.c~=,=2 :;~'= o.:~~Eii-f=-:..c. ~-=.. ct_---",

.....""

'"

c;¡

~ i:~ B~ IICo ,... II; :~1

...- '1C".. :i

t~ =~- .~ cs " :) ... ....r. .. 0... "'.. ~;¡ ~ E ~.. ;j :: ::

M ~~"i

4J

...i"":l

ë ¡;" ~:: ..~" '1.. .... :::: c.. 0~ õ= a.;; '1": C.. ..~ """ !ë '1~ "

E~

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~.... ;:

-= ~§

c;¡

"" ~~ '1.. C~ ....Jl "ic v" ii'. .... ::c ~=; "-; ..E ::.. .~.. :..."..Õ

.i..c"""ii~

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1i ~ ~ i:,. -:I..;.:~c.&l '" : . s~~.. ~.i.: ~ r. X;:... 1i1-.: 7. : ;:..:=-=~t = --~ :: : ";

:i c:

~ ;:- "-c. :;.r. ~

~i

§;;..-N" ::

.. '; E..- .."..2~ ~ "i- ..., -

~~- c.

c( ;.

...- "c -.. ~"" -_ ""~ ~ ~~ s !~ ::-~ ~- J~ ,. ..B ~ ~.. ~-_.- :;

.. A'. ..:l :l '&

~ c ~

~ ~ l: sc _ - ~

.; ~ t ~;;=E~

44 J. R. Babb et ai.

plate5, Each diluted and undiluted sample was plated in duplicate as aprecaution against contamination or error, thus giving two sets of plate5from each hand.

:\11 plates were incubated at 37"C for 48 h. Colony forming units (cfu)were enumerated on all plates showing 30-300 colonies and the arithmeticmean of the duplicate plates was taken and, after making the necessary

allowance for the step in the serial dilution, the counts transposed into !ogio

\alues. The mean of these values was calculated for each hdnd and this is thebaseline count from which the post-treatment counts were subtracted todetermine effcacy.

Test formulations and study designIn study I the four surgical scrubs containing -l% chlorhexidine gluconate

were as follows: 'Chlorohex' (Geist1ich Pharmaceuticals, Switzerland);

'Hibiscrub' (lCI Pharmaceuticals, L'K); 'Cniscrub' (Seton Healthcare

Group, lK); 'Wicamclor' (Laboratories Wicam S:\, Spain).In study 2 the following formulations were compared: unmedicated tablet

soap, Type B (standard) (~imbus Laboratories Ltd., lK) 8S 1914,~ational Health Suppliers code :\IRA 560; 70% isopropanol (lPA) 30 sand

2 min application; 7,5% povidone-iodine, 'Betadine' Surgical scrub (~appLaboratories Ltd., CK); 2% triclosan skin cleanser, 'Aquasept (HoughHoseason & Co. Ltd., L'K).

Twenty-se\'en volunteers took part in study 1, and H in study 2. The testformulations were randomly allocated to each volunteer, on a párticular day,using a computer-generated randomization scheme, such that eachvolunteer used each formulation once only. The design is such that at least7 days elapse between each application to ensure that no persistent effectremains.

:\t the start of the study, immediate or prolonged treatments were

randomly allocated to the right or left hand of each volunteer, and thereafterthe same hand was used for all test preparations. A chart was prepared toindicate the volunteers' names, dates and times of arrival, scheme ofproducts to be used and hand allocation. During the week following thebaseline period, but not less than 48 h after the last baseline sampling, one of

the test treatments was examined for immediate and prolonged effect. Insubsequent weeks, 7 days were allowed to elapse before a further test wascarried out.

.\.ethod of application of test preparationsThe following procedure was used for all formulations. The hands werewashed for 30 s with unmedicated bar soap and the nails cleaned with amanicure stick under running water. Excess water was shaken off (aqueous

formulations) or the hands dried on paper towels (alcohol rubs) and 5 ml ofthe test formulation applied to the hands and forearms. The nails of eachhand were scrubbed for 15 s using a sterile nail brush and the hands and

Evaluation or iur¡icai band disinfection 4S

iny forming units (cfu)nies and the arithmeticmaking the necessary

its transposed into logioach hand and this IS thents were subtracted to

forearms washed for a further ISs and rinsed for 15 s with the fingertipsuppermost so that the water Rowed away from the clean hands A further5 ml of the product was applied and the hands and forearms washed, using astandard technique; four times in 2 min. Hands and forearms were finallyrinsed for 15 s and dried thoroughly using separate paper towels for handsand forearms. \\"here alcohol was use, additional aliquots were supplied as

it evaporates rapidly .\11 hands were dry before sampling.

,Iated in duplicate as a

:\ing two sets of plates

hlorhexidine gluconate

euticals, Switzerland):

ib' (Seton Healthcare

, Spain).ed: unmedicated tabletLtd., LK) BS 19H,ropanol (I P.\) 30 sandSurgical scrub ("appr. '.\quasept (Hough

Sampling procedUTt

The hand designated to determine the immediate effect of the treatment wassampled after washing, using the glove massaging technique described

under baseline counts. A sterile, unpowdered, loose.fitting surgical glovewas worn on the other hand for 3 h, during which time the volunteer wasfree to perform normal duties. At the endof the 3 h period, the gloved handwas sampled as before. Aliquots of both glove samples were diluted andplated as for the baseline count.

24- in study 2. The tester, on a particular day,me. such that each~n is such that at leastat no persistent effect

Calculation of effcacyThe effcacy of the products was assessed by measuring the reduction in theviable count of the resident skin flora. The immediate and prolonged effectswere calculated for each volunteer. by subtracting the 10gio count after

washing; from the appropriate mean logio baseline count. The mean logioimmediate and prolonged reductions were determined for each product (seeFigure 1). Each volunteer was his or her own control. It is conventional inthis type of study to work in logliJ units, so the mean counts ..ere geometricrather than arithmetic. Statistical comparisons were made using the Student't' .test between means of paired samples, for immediate and prolongedeffect.

ged treatments \\ereunteer, and thereafter.hart was prepared tof arri\'al. scheme ofe week following the=líne sampling, one of

prolonged effect. Inire a further test \\as

User acceptabilityThe four chlorhexidine products were presented in similar containers andcoded so that volunteers did not recognize the product used.

During the first study, in which four chlorhexidine scrubs were

evaluated, volunteers were asked to comment on the cosmetic, or userfriendly, qualities of the products. They \i"ere asked to give ratings of good

(+ 1). moderate (which included no comment 0) or poor (- 1) for thequalities of feel, perfume and lathering.

.ns. The hands werenails cleaned with a, shaken off (aqueoushoI rubs) and 5 ml of

is. The nails of each

1 and the hands and

Results

The immediate and prolonged effect of applying four chlorhexidineformulations is sho\l n in Table I. .-\lthough the test formulations were allreported as having the same active ingredient, i.e. 4% chlorhexidinegluconate, the differences between formulations varied considerably.

'Hibiscrub' (lei Pharmaceuticals) gave the best immediate and prolonged

4ó J. R, Babb et ai.

Tibl~ i. A H~ii'"~"t of flu ~fJ(O(Y offou' (JilorJio:idl"~ su'gicoi scrubs

\Iean log,. reduction in resident ikin 110ri----~--(mm~dilte eff~ct Prolong~d effect

(3 h)-----------'Wicamclor''Chlorohu''Hibiscrub''lniscrub'

0.) 5

00481010804

0.110.)7, 16

0.97

E\ ¡IUiltiOn c.rri~ out on 27 ..olunrren

effect with 10g10 reductìons of 1.01 for immediate and 1.16 for prolongedeffect. There was no significant difference between 'Hibiscrub' and'Cniscrub' (Seton Healthcare) but both gave significantly better resultsthan 'Chlorohex' (Geistlich Pharmaceuticals) and 'Wicamclor'

(Laboratories Wicam SA).The results of user acceptability assessment are shown in Table I I. The

most popular product overall "as 'Chlorohex', closely followed by'Hibiscrub'. 'L'niscrub' was the least popular.

The results of the second study are shown in Table IlL. The mean logio

reductions for immediate and prolonged effect showed that a 2 minapplication of 70% IPA was the most effective of the treatments, givinglogio reductions of i .65 for immediate effect and 1,58 for prolonged effect. A30 s application of 70% IPA was almost as effective, gi\'ing logio reductionsof i .50 and t .H; 'Betadine' gave a 10gio reduction of 0.80 for the immediateeffect which was similar to the more effective chlorhexidine scrubs. i.e.'Hibiscrub' t.O 1 and 'C niscrub' 0.8-l shown in Table i. The prolongedeffect was poor, however, giving a log,o reduction of 0-43. 'Aquasept, wasthe least effecti\'e antiseptic formulation in the immediate test (0' 29) with aprolonged effect similar to that of 'Betadine' (0,4-9). ~o reduction wasachieved using unmedicated bar soap, There were no significant differencesbetween the immediate or prolonged effect of a 30 sand 2 min application of70% IPA. The alcohols were significantly more effective than 'Betadine',

Tabl~ II. ..hs~ssml't of ,~sponst: (osm~tlc Qruptability

Rank ord~r of prl.f~r~nc~

'Wicamc1or' 'Chlorohu' 'Hibiscrub' 'L'niscrub'

Feel (Ii) 2 (041'3 (12) I (0) 4

Perfume (7) 2 (I i) i (2) 3 (- 6) 4

Lather (7) 3 (22) i (\6) 2 (- 10) 4

O,'er.1I preference (25) 3 (37) i (30) 2 (-16) 4

Score: goo + 1. moerate O. por - 1. I -- Iù...ui order of dJectì\ entu.

Evaluation or surgical band disinfection 47

. 'ridirr~ ¡"'gual icr"bi Tabl~ II i. AIUiimtrrl of IJu tjJe¡Je) of ,,,'gieal Jiarrd diilrrfutlO' p,odlJ

'sid~nt sk,n Aor.\It.n log,., rtduction in rt"d"nr ,kin Aor.

Prolongtd ~ff~ci(3 h)

0.\10.371160.97

I mm~diar~ effect Prolong~d ~ffC'i(J h)

lnm~dicattd ,oap2% Triclos;n cl~ans~r ('.\quasepl)7 5'1. Pondone scrub ('B~iadin~')70"0 I P.\ r 30 s .pplication)700; I P.\ (2 min applicaiion)

-005029080I 50

165

-009049043I HI 58

E\ aluation cirrltd out using ~4 '" olunr~n.

e and i. i 6 for prolongedetween 'Hibiscrub' andgnificantly better resultsiIs) and '\\ïcamelor'

able IlL. The mean Jogia

: showed that a 2 min.f the treatments, giving.8 for prolonged effect. A~. giving Jogia reductions,f 0.80 for the immediatei10rhexidine scrubs, i.e.rable I. The prolonged

of 0-43. '.-quasept, was

iediate test (0'29) with a

-l9). ~o reduction was10 significant differences

and 2 min application offective than 'Betadine',

'Aquasept and unmedicated soap for both immediate and prolonged effect.'Betadine' was significantly more effective than '.\quasept andunmedicated soap for immediate effect and 'Aquasept was significantlymore effeciive than unmedicated soap for the immediate and prolonged

effect. There was no significant difference between 'Aquasept and'Betadine' for prolonged effect. but both were more effective thanunmedicated soap.

.\lthough the formulations in studies i and 2 cannot be compared

statistically, the mean logio reduction factor -RF) for 'Hibiscrub' wouldappear to lie between the alcohols and 'Betadine' for both immediate andprolonged effect.

shown in Table I I. The,', closely followed by

Discussion

12) 1

(2) 316) 2

JO) 2

(0)0(-6H

(-10) ~(- 16) ..

The glove washing test used in this study is more ïealistic than tests inwhich a bowl,l: petri dish') or plastic bag'l is used for sampling. It alsoallows volunteers to continue working during the period required formeasuring the prolonged or persistent effect. The time of 3 h chosen formeasuring the prolonged effect is a compromise; most operations will becompleted in less than 3 h. The test is repeatable and reproducible as

demonstrated in a study carried out by four laboratories.aThe present study has extended the use of the test to additional products.

The results of study i demonstrate the need for such a test, sinceformulations with a similar concentration of the same acti\.e agent variedconsiderably. The logio reductions in counts following applications ofdifferent formulations of -l% chlorhexidine gluconate ranged from 0.35 to

i.o i when sampled immediately and from 0.1 i to i. i 6 when sampled after3 h.

The accèptability of a product to staff is as important as its antibacterialactivity.

IS An unpleasant or irritant preparation will not be used and damage

to the sl.in may be associated with increased bacterial colonization of thetreated hands. .\Iso, poor lathering will encourage the use of a second

aliquot of soap or detergent, and this would double the cost. The four

eptabi1ity

er~nce

biscrub' 'lniscrub'

48 J. R. Babb el aJ.

chlorhexidine products sho..ed considerable differences in acceptability,but a longer trial is required to assess skin damage.

In study 2 other qmimonly used agents were included and compared withnon-medicated soap. The results were similar to pre\'ious studies usingother methods of sampling.

i

Time iS important to the surgeon and reducing scrub time without harmto patients is ob\ iously desirable. In the past, the surgical scrub consisted of

scrubbing the hands and forearms with a brush for 10 min, often followedby immersion in alcohoL. Similar results are now obtained with a 2 minapplication of an antiseptic preparation without scrubbing.

In the present study a similar logio reduction was obtained by a 30 s as bya 2 min application of 70% IP.\. This is the same application time as

commonly used in the hygienic hand disinfection test.s A short applicationperiod of alcohol was also suggested by Selwyn & Ellis.'6

All products showed a small login reduction in this test as compared ..ithtests measuring the removal or killing of transient organisms. Even after a2 min application of the most effective product. IOJ organisms were released

from the hands In addition. many of the superficial residents and transientswill ha\'e been rerno\.ed by the preliminary rinse with water. A longerapplication (5 mins) of n-propanol or chlorhexidine detergent followed byalcoholic chl0rhexidine (4 min application) showed logio reductions of 2.5for the immediate and I'ïl for the sustained effect." \\"hether prolongedapplication is of \-alue remains uncertain.

If glo\.es remain intact, disinfection of the hands is not required.

Howe\.er, glo\.es are frequently punctured during an operation and

organisms may escape into the wound. iS The numbers of these organismswill be reduced by the application of an appropriate disinfectant.particularly if there is a good persistent effect. The inRuence of glove

punctures on the incidence of wound infection remains uncertain.IQ.2o butthe potential hazard of infection from the resident Rora of the surgeon is

likely to be greater with some operations, e.g. insertion of a hip prosthesis.Since the emergence of HI\' infection the glove is now seen to be more as aprotection to the surgeon than to the patient.i ~ew techniques in handlingsharps, double gloving and new glo\"~ materials are likely to reduce glovepunctures during surgical operations in the future.

.\s already discussed with hygienic hand disinfection, the interpretationof results in terms of clinical effect is not possible. ~o convincing controlledclinical trials ha\.e been reported and statistical differences in laboratorytests do not necessarily imply differences in infection risk.22 However,

despite these uncertainties and limited effect of disinfection on the residentRora, it is likely that tests for surgical hand disinfection will continue to berequired. A standard test is necessary and the test described in this paper issuffciently reproducible to fulfil this role. Products can be placed in orderof effectiveness and the value of different methods of application assessed.

The user must decide on the most appropriate product after taking allaspects. including user acceptability, into consideration.

differences in acCeptdtiil¡(~.,

mage.included and compared ii Ithir to pre\ious studies using

Evalu~tion o( iureical hand d.iin!~tiOQ49

Rererenc~

'ction, the interpretation'0 com-incing controlled

lifferences in laborator\"

ection risk. ~~ Ho\\e\e~,

nfection on the resident.tion will Continue to beescribed in this paper is; can be placed in orderof application assessed.

.roduct after taking alltion.

I. .i.\.iirf~ G.-\J The effe't of mlibacterial agents on ih~ Aora of th~ skin. J Hoip ¡"j~((I 980: I II i -I H

2. Re\brouck G Hand\'Ishlng and hand disinf~ction J floip 1"lul 1986; 8: ;-23Rott~r \1 Pondone'iodine and chlorhexidin~ gluconat~ containing d~iergents fordisinf~ction of hands J ii",p ¡"jut i 981. 2: 2iJ-276.

4 Price PB. The bacteriolog\ of normal skin a n~" quantitatl\~ t~st appli~d to a studi ofthe bacterial tiora and the disinfectant action of m~chanical ci~ansing. J ¡"lui DIl 1938;6J: 301-318

5. Lo," bury EJ L. Lilly H .-\. D"infection of rhe hands of surg~ons and nurs~s 8,\f~d J1960: i IH;-1450

6. Rotter \1. \lltterma\~r H. "undi \1 lnrersuchung~n zum modell d~r kuntslichkontaminierten Hand -\.orschlag für eine Prüfmethod~ Znitralbl 8akt~"ol flyg .-lbtO"g, R~,h~ 81974: 159 560381

i. .-\yliffe G.i.J, Babb JR. Quoraishi-\ H. .-\ test for 'hi gi~nic' hand disinfection J Cli"Pathol i 978; J I. 923 -928

8. .-\yliff~ G.i.J. Babb JR. Oa\ies JG. '~\\som S\\, Ro\\land C. Platt JH, \lason BHygienic hand disinf~ction rest s in :hr~~ laborarori~s J Hosp 1"lut 1990; 16: 14 i -149

9. Hollo\\a\ P\1. Platt JH. R~\brouèk G. L.illi H.i.. \Iehtar S. Drabu Y. :\ muiticentre~i.aluati~n of t\\O chiorhe\id;n~ containing fo'rniulations for surgical hand disinf~Ç(lon JHosp ¡"IUI 1990; 16: 151-159

10. P~terson .i.F. Th~ microbiolog\ of the hands. In: Drt~Iop"u"ts i" ¡"dustrial.\ficrobiology, 14. \\ash,ngton DC. .-\merican (nstiiut~ of Biological Scienc~s i 973;

125-130.11. \ 1 ichaud HS, \ (cGral h \ lB. Goss \\.\. i mpro\td experimental model for measuring

skin d~g"rming actinty on rh~ human hand .-lltimicrob .-lg~"ts Chnriothn 1972; 2: 8-1512. Lowburv EJ L. Lilly H.\ l ~ of four per cent chlorht'x,d,ne d~t~rgent solution

(Hibiscrubi and other meih.--es of skin d,sinfection. B, .\f,d J 1973; 1: 510-515.i 3. Rotter \1. "oller \\. \\e\\alka G. \\t'rnu HP. .i.ylitf~ G.i.J. Babb JR. E\'luation of

procedures ior h~.git'nic hand disinft'ction: controlled parallel t'x~riments on the \'i~nnat"si modt'. J Hye 'Ca",bridg~ 1986: 96: 27-37.

14. Gas~-ht'n \1. Etude .:omparali\t' de d~ux rn~ihod~s destin~~s au pre\'lement de la Aor~

microbienn~ des mains Htit Ch"u'g . leta 1968; 35: 3i2-3i7.15. Ayliife G.i.J, Babb JR. Da\ies JG. Lilly H.\. Hand d,sinfeition: A comparison ofvarious agents in laborarory and \\ard srudj~s. J Hasp 1"lul 1988; 11: 226-243.

I fl. Sei" yn :-. Ellis H. 5kin bacteria and skin disinfe-tion rt'considt'rt'd. B, .Htd J 1972; 1:136-1 -W.

17. Rotter :\1. Koller \\'. Sur~ical hand disinfection: effect of ~quential u~ of t"ochlorhexidint' preparations. J flosp ¡"Itet 1990; :6: 161-166.

18. Oodds RD.i.. Guys PJ. Peacock .\\1. Duffy SR. Barker SGE, Thomas ~fH. Surgicalglo\'e perforation. 8, J Su,l 1988; 75: 966-968.

19. Cruse P J E, Foord R. .i. ñ\.e year prospectl\~ siudy of 23649 surgical wounds. A,ch Su,l1973; 107: 206-~10.

~o Crust' PJ E. Foord R. Tht' c:pidemiolog\ of ,"ound inft'ction. .i. 10 yur pros~ctive studyof 62939 \\ounds. Symposium of sur¡¡lCallnfectlons. Su'g Cilii '11 .\o,th .4"'('i('a i 980;60: 27-4.

21 Hus~ain 5.\. Llllf .\B.\. Ch'~udhary .\.\.\.\. Risk to surgeons: .i. sur".~y of accidentalinjuries during operatIons. 8, J Su,l i 98lì; 75: 31 +-3 i 6. _

" Ayliife G.\l. Standardization oi disinfectant testing J Hosp Iiilut 1989; 13: 211-216.

ng scrub time \1 ithout harmie surgical scrub consisted ofh for 10 min, often follo\\ed

now obtained \I Ith a 2 mint scrubbing\\as obtained b~' a 30 s as by

~ same application time asin test.~ ;\ short application& Eiiis.l~

i this test as compared with'nt organisms. Eien after aoj organisms \\ere released'ial residents and transientsnse with \later. .\ longerline detergent followed by. ed log i" reductions of 2.5fect.'; \\'hether prolunged

hands is not required.uring an operation andmbers of these organismsappropriate disinfectant,

The inHuence of glo\e~mains uncertain, 14~" but'It flora of the surgeon is'nion of a hip prosthesis.no\\ seen to be more as a1\ techniques in handlingre likely to reduce glo\'e

î~'~~~~~~~~Ej;i~\~tfi~01.~tj~W~t~t~tØ:¿~£~!~~~:"-?:,~;;~,.. : ;~' ."~""'\1 '~''s,.~~i:'~:6o~~;Q ~-.!.. .,.,..1.. ,~~;¥" ~¿,"t~'.' ~-., "'f..q~-:_;'.l.'t' ~~P., ,,- ~'''~~i-:~'''_-/,, .02 ':' ,"7,- c.... - .. _,;- 'm "" ~~" ~ 'C.-..,.... "'. . '.-""Y; . y/....:t',;d~..'.. ""-..~~ ."~- ",'~' ~ ." _~. ~ _~. . 'f _, /0. '. _'. .." u:\ '1 :t:;lr_;~~o.~~i.:;., ~~. "'- ...J.)..~. ~ '-'"1~;- \.'.__"."''~":-;'"1 '""....-.,.l~ :,.-~:-,",, ,...:'"i-~~ .... _ -i.C'i'~,t~ . ,4""1 ~'~_'.' ~.,. _. ....._ .;_.... ~ .. .. -:-' ,Jj' ,L. ~. .'.:, "P.,- -.~' .-;.' "O.,."t - i.-"'~.i.. ....T..\..". '- ~~.; - 'r.-""~~ _,,, _-_~._ 0i. .' _.~ ò. ~ I. ..- ¡;. ;¡i~.~~... ::. ;~,;;. :,- ._,~.~,'S~.,;.:.~,, ~,'1,,"'-'-;'''~.. .".,...::t.......r-~, .:_ '~'3';."'';-)¿._.'-". _ ,.,,;::. ;,.' _... ~ 'q'. ,..~.., .,. 'fA.... '~l i!r.t-.. ..- ..... -'.. ~. .:'~..l. -' ~c:~'-.' ~ if~..,. _~~.... "l. ~'-"':"-i' ~-I._ '.. ~""" ,_ _; .

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-bioI. 10:279_

Nov f),95 liT:53 No .005 F .O¿

lVol. iJ. No.4'

Increased Bactericidal Activity of Dilute Preparations ofPovidone-Iodine Solutions

Runi 1.. BERKfLMAN,. BETTY W. HOLL.AND. M"O ROGER L.. ANDERSON

Høspiiall'l"iloiii Pro,røfl. Clflltr 10' lriuliOUJ Diuo1t. CtlttrJ for DiSflUt COlii,ol, M/øflia, Cito'ría

JOJJJ

nliiaiive. 5Co

ni 'ssay ror

;94299.'r. ui" R. J.

FOlin phenol

'. ManUlI of,Iiciiion no,

~nd Weirue.:'irol. Aila

'. Mic,oh_e-

., inlibon

.:ia"meni or

IIh Sci.ic..

Received 15 Ociober 19811Accepied I December 1981

Recent conftrmation of intrnsic bacierial coniaminaiion of 10% povidone.iodine solution has raised questions re¡arding the baciericidal mechanism o(iodophors and the possibility for survival of veieiaii"'e bacterial cells in iodophorsolutions. In this laboratory invesiii:ation, /lve ditrereni species were exposed tovarousdiluiions of three commercial preparations o( 10% povidone-iodine

solution¡ survival was assessed after exposure (or time periods varying between 0and 8 min. All brands or povidone.iodine solution testd demonstrated more rapidkilling of SlapJiiloc()CCUS aI/reus andMycobaCltf¡um cJie/onti at dilutions of 1:2.1:4,1:10, 1:.s0, and 1:100 thar did the siock solutions. S, aurtliS survived a 2.mineJposure to (ull.sten¡th povidone.iodine solution but did not survive a i.s.$exposure to a 1 :100 dilution of thc iodophor, BOih stock and dihlte preparaiions o(10% povidone.iodine solution demonslraicd rapid baCtercidal aciion aiaiiisiKltbsiel/a pntumonict, Psiudomonai ctpac/a, and Str,piocoCCIiS mllis,

'11"1, inc J,, or ELlS..'crodi.,no.ikon. Ned.

'."'!, anddiaino51ic

'1 (or 1"6,

iii. 1"1.

:CS. F£85

rion orlhein ihl l~

.:'~JI.ilell. 1975~'ror Ch..

Iodine has been recoinized as a valuable

antiseptic for more lhan a century. Howiver,iodine bums, caused mainly by a 7% tincture.iave iodine a bad repuiation ($). In the 1950s,iodophors,loose complexes of iodine and acarrier, were formulaied to eliminaie the sideeffects or iodine while retaining iodine's bacil'-¡'cidaJ activiiy (H. A. Shelanski, U.S. patent2,739,922, March 1956). lodophors have nowbeen used in clinical praciice (or more than 20years; povidone'iodine, the common name (orIodine in complex with the nonsurfactani carrerpolyvinylpyrrolidone, is currently the mostWidely used iodophor (5). These prepiuiuionsenjoy widespread popularty today, attrbutable

to their lack of skin irrtaiion and their absenceof odor and sllinini. More important, only a (ewinvesligaiors hive questioned their e6'cacy (7,9). However, recent con/\mation of intrinsicContamination or a 10% p~vidone.iodine solu.

\jon with Priiidonio"ar ttpaciø starilcd manymicrobiòlogists and çhcmists considered expertsin the antisepiic: and disinfectant field (1. 2).Althou¡h mechanical protection of P. ClPQciøby organic or inorganic: mallrial is no.. consid.

ered the most likely explanaiion ror the findini.debate over the mecharism of the solu iion' sbactericidal activity has coniinued.

In thc work reported here, we studied thebaciericidal activiiy of povidonc-iodine by chal.Ic:ngini: diferent species of bacteria to vllousconCentrations of povidone-iodine solution. Theresults have ma.jor implications both theoretical.ly, in further dc:finini the possible bactericidal

15177. USl:.IS..' ilnd\opl..mo.

NOV-Ø6-1995 09: 59 121219435653

mechanisms or povidone.iodine preparations,and praCtically, in the use of povidone.iodine bypaiieni.care proiiiders,

MATERlA ,\O METHODSPoVldont.iodint soluUoni u..d wtlh chall.nie midiu.

Three 10% povidone. iodine soluiions were used: Pharo

madine, 101 X80299 (Sherwood Pharmaceutical Co..Mahwah, N.J.); Beudìnc, loc A09 (Purdue Fredenek.Norwalk. Conn.); and Povidine, IOl OLS09 (NiûonaJ

PharmactuiicaJ Manu(aeiurin¡ Co" Baltimore. Md.),Ten milililen offull-itenSlh povidone.iodine and i :2.1:04,1:10.1:'0.1:100.1:1.00. and 1:10.00

dilutions otClch povidone. iodine solution Were slparaiely added10 16 by I'-.rum scre...cap tubes. The dilueni wis

sterle deioniied waler. The pH or each ieu diluiionand sioell soluiion was measured.

Chlllcn" bicieria and Inociilym size. Five di~erent

ba.ierial species were u3ed in these povidone.iodineehallen,e siudies. One strn each of SlaphyløcøWu,,¡reus (ATCe 159m, Kieiniillø piieiii"oiiiot (a elini.ca isollle), 1'. cepac/a (isolated from Pharadine. lotX8019), and S"'PIOCøccus fIirls (¡soiiied from ihuman ihroat) "'as ,rown sepuatoly in 5 ml of brainliell infusion broin (DitO Laboraiones. Deiroh.Mic.,) ror iI 10 204 h ii 37'C:i MycClbac"riufl

Cht/OMi main (M3. 110laied (rom a porcine heanval..e remnanllwas irow" in 7 ml of? H9 broih (Difco)for 71 10 96 h II 22'C. All iubes 01 brolh ..ere

cenirifuged ror 20 min ai 2.00 'X i in a Sorvall (IvanSorvall Inc., Newtown. Conn,) an,le cenlriru'¡e. iheliquid phaie Wa, dìie~rded. and the pellci "'Ii. ius-pended in 5 ml or O.OOJ M phosphaic.bullered walcr(PBW) of pH 7.2, Each baeteri,) suipension wasIhorOulhly mi'id: I ml was removed and added 10 a 9.ml PBW dilution blank. These 1:10 dilu\Ìons Ihenbecame the siock inocula; 1 ml oflhe 1 :10 dilution was

63

96% P.02

. I

NOU-0S-1995 10: 00

. i. L-' .i L. lj .. ;J.., .vì .J U _ì ..

636 BERKElMAN. HOllAND. AND ANDERSON

ì, U v (" ': =' L II . ) _~ ì\i 0 . U 1.1:' .

TABLE 1, E~eci of COncen:raiion of povidone.iodine Soluiioni and length of exposuie on Ihe survival or$..QurruJ

J. eii!", MICiioaIOl.

Ililulions pHNo. or orianiimi reco.credlO,lml' an.. .0nl",1 iim.'

Ij i '0 i I min ~ "'in , minPhamiadine

Slock,lO% :U 1:2 20 1 0 01:2 3.3 98 3 0 0 01:4 U 67 0 0 0 01:10 3.7 0 0 0 0 01:50 4,0 0 0 0 0 01:100 4. 0 0 0 0 01:1.00 5.9 340 38 1 0 01:10.00 6." ;:l01 ;:i 01 ;:io ~IO) ~ioiBeiadineSiock. 10% l.a ::lO 508 125 6 01:2 l.7 443 51 I 0 01:4 3. 160 I 0 0 01:10 U 0 0 0 0 01:$0 4.1 0 0 0 0 01:100 4.7 0 0 0 0 01:1.00 5.7 43 2 0 0 01:10.00 6,7 ::101 :. 101 ~IOI ::10 ::101ProvjdineStock, 109 3. :0101 192 3a 1 01:2 U ).0 42 0 0 01:4 ),6 26 0 0 0 01:10 J. 0 0 0 0 01:50 4,0 0 0 0 0 01:100 U 0 0 0 0 01:1.00 5. 7 1 0 0 01:10.00 15. :010' ::105 :0 10J ::10' ::10-\· Represenu 0.1 ml of. 1:10 diluiion Cl ml of iesi poYidone,iodine and 9 ml o( 0.003 M eonlaininI0,5Ç\

sodium Ih¡osul(,IC).· lOO survivil · 1.7 l( 10' orpnisms/O, I ml.

Idded 10 each of the three brands o( poYidone.iodineat e"h of the iest eoncentrailons IStQ(k and seuen

diluiions).To deltnnine Ihe number of orianisms per milliliier

o( inoculum. 1 ml of each siock inoculum "'IS diJuied(10.1 1010.') in 9.mJ paw dilution blanks, In dupli-cate, 0.1 mJ was removed (rom ihe 10-1 10 10'.diluiion iulls and separately plaled on Tryplic81~ soy

aiar (181. Mierobioloiy Syuems, CockeysYile. Md,)"'iih ""sheep eryihroytes (1$A8); each portion wasspread over Ihe .,ir surface with sierile bent,slassrod., and TS"8 pl.I" were incubated IS pre~iously

describtd ror broth '''Ilures. Cilculaied i!loculi permilililtr were as (ollows: S. ØClrtuS. 1.7 )( 10'; K.

P"III"'Olilot. 1.0 )(10'; l';r,pø(j(J. 2.9 l( 10': S. "'¡';i,

U l( 10': an M. r1i,/olii/. 9.6 )( lot.Simpllni,nd dtlirmlnailon of biciericidlI ,!riel or

po'idon..ludln, SOluilol\, Samples were r~moyed (rompovidone.iodine soluiions It 1$ i!ld 30 s snd at 1.2.4.and' min (or subsequcnt deierminalion or surviviniorpnisms. ...1 each sampling iime. 1.0 ml or ihepovidone.iodine suspension 10 be leHed wu removed'!lei added to a 9-ml PBW diluiion bl~nk coniainin,O,5'7ø sodium ihiosuir~ie (10 neuiralize residual iodini),

The 0.5% sodium IhiolUlralc soluiion was previousiytesled and (ound noi 10 be inhibitory to any of Ihech'llen,c orianisms. E~eh iube was thoroughly

12019435653

mixed: 0.1 ml wis lhen removed in duplicate andadded 10 the surface or TSA8 plain, Tesi ponionswere ,venly spread ouer the eSal surface Wiih indiyid.uil tlerile bcni.alass rods. TSAB plaies inoculaicdwiili poYidone.iodine eoniainini S, ,limn. K. P"''''

"'øriù:i. 1'. cepollo, and S. "';1;1 were incubated ai37'C (or 24 to 4. h; TSAB plales 10 which M, rli,/a",;wu inoculated were incubiied al 22'C ror , dayi,Mer inciibiiiòn. colonies present On TSAB plaieiwue enumeraied ind recorded per 0, I ml

Dcl"minilioii of ivill.blt lodlnc. Au&ilable iodine

concenirations or siock povidone.iodine soluiions indIhree iest diluiions (\:2.1:4. ind 1:10) were perlomiedby thiosulfate iilraiion II I commercii

I laborliol'Y(MacMillan Research, Maneiia. Oa,).

RESULTSAll brands of povidone.iodine solution demo

onsiated more rapid kil of S. ai"ius and M.chelonei at dilutions o( 1:2. 1:4. 1:10, 1:50. and

1:100 than did the stock soluiions (Tables 1 and

2). In most instances. the 1:1,00 dilution killedS. allrta and M. che/onei as rapidly as did ihc

(ull.strength solution. Figures 1 ¡lid 2 sholl' ihee6ccts of po..idonc.iodinc in various concentra.

95%

YOL, 1'. 198i

TABLE 2 Eo.

I Diluiion

I

P\adincSloclr.I~1:1

II:~1:10

I1:01:100

i1:1.001:10,00

I Bcl.dine, Stock. 1O'I 1:2

J

1:41:101:50

l

1:1001:1.001:10.00

Po..idineStoek,lO%1:21:41:101:501:1001:1.001:10,00

. Sce Table

· 100 iur,

)

j

iions on thelollt;. respe

Both stoepovidone.ioaciion agair

S. mitis. Ti

from eitheras high as Iwere they r1:1,00 dilisoluiions, idcmonstraisure or Ihc

The avaidone.iodin.10,00 "",ilIe~ree of c

dilutions r-

PrCVlOUCOnccnira'cidal after

Ihc ~olutilstudies ha

t.iions 0

P,Ø3

...0:

ooooooo

:'lO .

ooooooo

:.10'

ooooooo

:'10'

iinin. O.SÇf

;ilicaie ind'II ponionsiill indjvid.inocul3lcil

" X. pn~ii.:eubal~d ~iII. ch"olltl'or f dars,¡AS plaies

.ble iodinr!uiioni ~nd

performcd¡aboraiol''

ion demo

; and ,\/.1:50, andles 1 andon kileds did Ihe

¡how th~'ncenira.

. ....' ., '" v ., ':j -+ -. .. U .' .J

VOL. IS, 1982

I~OV Q,';) 1U:')) NO.Uü') ~ ,..J

..CTIVITY OF' POVIDONE.IODINE SOLUTIONS

TABLE i. E~eci of concenlralion of povidone. iodine ~oluiion\ and len~ili or e:iposure on ihe survi\al of

M, ch,ioii,;

637

4-; Diluiio.. No. or or,ani¡mi rtcovertd-I. i ml' .ntr Conl.,1 limc'--IS i )0 i i min ~ "'i" 4 min I minPnarmadineSlock,lO% :.10J :'101 ::IOl ::10 160 01:2 )0101 ::101 ::ia 390 0 01:4 )o~ )oW :.10 0 0 01:/0 ;) 0' 466 0 0 0 01:50 ::10' 10 0 0 0 01:100 )oløJ . 0 0 0 01:1,00 ::10" ::W )oIOl i 0 01:/0.00 )010' ;)101 :;10' ::10" :;10 :. i ojBeiadineSlock.IO% ::10) ::101 ;:10) ::10l :'10' 01:2 :;10' :;10' ::IOl ::10' IS 01:4 :.10' :;10' )oIOl 78 10 01:10 ;)10' ::10) ::10' 0 0 01:50 ::10 J2j 1 0 0 01:100 ::løJ 16 0 0 0 01:1,00 ::10' ::101 )o10l 32 1 01:10.00 ::10' :OlOl )0 101 ::10) ::IOl :oWI'ovidine

Sio~k. 104 :0101 ::10) ::10' :oio 150 0i:i :'W ::IOl ::io =-w 0 01:4 =-10) :'10' ::10) 49 0 01:10 :.10 :;10' 104 0 0 01:50 ::10 J2 0 0 0 01:100 )oW 0 0 0 0 01:1.00 ::10' :.IOl 184 0 0 01:10.00 :.10' :.IOJ ::10) ::JO ::10' ::lOl· See Table I.· 100% survival = 9.6 )( 10' orøanisrnslO.I mi.

)

,

iions on the survivaJ of S. al/flllS and M. eli"/o"f.i, respectively.

Both stock solution and diluie preparaiions ofPOvidone-iodine demcinslraled rapid bactericid~1aclion against K. p"tHmoliiul!. P. cepacia. andS. miiis. These organisms were noi recoveredfrom eiiher Ihe ru/l.sirellth solution or dilulions

as high as 1:100 after a coniaCllime of is s. nor'Nere they reÇ(vered af1er exposure for 30 S 10 a1:1,00 dilulion of each of the povidone.iodinesolutions. However. no baciericidal action wasdemonstrated by a 1: 1 0,00 dilution afler upo.

Sure ot tho test orianisms fOr up to 8 min.The availabl. Iodin. content of all stock povi.

done.iodine SOlutions wuapproiiimaiely 19l or10.00 ¡te/tTl: it decreased

In proportion 10 Ihedeiree of dilution. The pH increised with hiiherdiluiions (Table 1),

DISCUSSIONPrevious studies have demonmaied thai low

i:nc:eniraiions of povidone.iodlne are bac:ieri.cJd~ aner Ve¡eiative organisms are exposed toihe SOIUlion for 24 to 48 h (6. 11). However. fewsiudies have assessed lhe e!feci of diluic coneen.ltalions of povjdone.iodin~ on the survival of

NOU-06-199S 10: 01 12019435653

bacteria after brief exposure iimes. L.acey re.poned enhanced bactericidal activity in lowconceniraiions (less ihan 0.1%) of povidonc-

iodine Solulions, but detailed experimenial datawere noi provided (8). Our investi¡:llion demon.siraied chai low concenirations (i.e., 0.1 to 1%)were more rapidly baciericidal ihen A full.strengih (i.e., 10%) solution. Indeed. in ourexpcrimenlS. a 1:100 diluiion of 10% povidone.iodine solution kiled bacteria "atleasi as quicklyas solutions of higher Concentraiion.

The chemiSlY of povidone.iodine is complexand not well undersiood. Therefore, the phe.

nomenon of inc:reased baciericidal activity withdilulion is diflculi 10 explain. Ceriainly, thisstudy establishes that lhe available or lhiosul.faic.tiiratable iodine. cUlTently the only roulinemeasurement or iodine comeni in povidone.iodine products, is not directly related 10 bacie.ricidal ¡ct¡viii' (Fia. 1 and 2). One hypothesis Isihallhe concenlrCliion of "fru" iodine (j,e" lhe

elemental iodine in solution) significantly COn.

tributes 10 the baciericidal activity of povidone.iodin~ soluiion (4). Tl'eman ha5 siated Ihndilution of povidon~.iodine results in weakeningof ihe iodine: linkage 10 lhe carrier polymer wiih

95%

'CoL.' ~LIJ~,:'-.):iti::.) Nov b,~~ 1U:53 NO.OOS FOS

638 BERKELMAN. HOLLA~D, AND ANDEkSON1. Cli'" MII:ROaloi VOl. 1.. I,

~3,0..ir;l~ 20~

ld

J 1.0

j

iodine le\,¡¡nous ec

h)'POihcsisThc res

imporianiManufaciuseptic purdine SO/Ul

period of

povidone.lions ma~iions is noincrease irwill inere

L Furihermrwiih frce

I orsanic diodine ma

I iodine:. Ir

solutionsbeen repiuses inch:preparaiicThese pn.and. wiih,

scarch isdilute pre

advocaieibranes.

Patienitwccn an:po,idoneuse as an

(or use 0eVer. in rman\' asaGen't r(,r

\. insiiiuiioI used Iibc

l boille to:

and othe

slrain of

I full.sirenfinding r, mendaii.

1 based or

en havL

before u'nelshouIhe oni~

study de

tion ma~iy,

PHARMADINE

BETAOINEPOVIOINE

-\~

\\\ \\ \\\\

" \\. \", \

", \',~"o 1,0 Ul 2.3 3,( 3.4 3.'7 4.0+

lli..oo (nOOa fUooi (l.~(:iO¡ 11'.lli ZL ('vlI'Sl/e'al~1Avoiloble rodine Conetniroloon (L.g 10 PPM)

FlO. I, SUr'ivlJ or s, aU'lUI aier e~pO$lJre (or 1 S s 10 povidone,iodine solulions o( various conceniraiioni

"Maiimum Su,.¡val of 1. l( 10. cells, ., Full-menllth povidone.iodine .. 10.00 ppm. 10,00.., of availableiodina ptr ml. Numberi in pa,eni~esei represent iiie diluiion of povidone'iodine.

a concomitant increase in the amount o( ~Iemen.ial ((ree) iodine in Soluiion (J 4). Tn the preseniinvestigaiion. we allempietJ 10 sUbstanliate ihisrise in (ree iodine with diluiion. lnde-ed, wiih then.heprane eiilrac;tion assay (M. W. Winicov andW. Schmii:t. L',S. paieni 3.028.299, April 1962;

A. Cantor and M. W. Winieov. U.S. patent

3.28.00, April 1962). levels of free iodine in.creased with dilution. However, this method ordeierminìn¡ (ree iodine is controversiaL. and ourdeterminations, particularly Ihose made ai high.er dilutions (i.e., sreiiier than 1:10) or IOr'povidone.iodine solution. were noi reproduc.ible. An aCCurate mc;ihod (or deierminin¡ (ret

I

I

í

,

~;"

#11

I:

/"

!i

- piHA~MAOIN£-- 8£iAOIN£.. . - -- POVIOINE

*¡

r;

l12.

li~J

1. 2.0 2,3 3.0 3.4 3.'7 4.0 .,(1:10,0001 11:ioool 11:ioo)(1 ~Ol ¡¡:JOI 11:41 U:2J Willi SI'cnQr~iAVOlloOle IOdi". ConeeM'olion (Looio PPM)

FlO. 2. SUT\iv¡1 of M. dulonti ~ftcr e~pOiure for 2 min 10 POvidone-iodine SOlulions or vlTious concenira.

iions. "Mò\imum IUr'ival of 9.6 lC IOJ cells, ~, Full.siren¡ih povidone.iodine .. 10,00 ppm" 10.00 ..1 orivailable iodine per ml, Numbers in parenlhue$ represent llle dilution of povidone-iodine.

NOU-Ø6-1995 1Ø:Ø1 12019435653

,. , (JG

,

.~o,

oneentraiions."" or aviilable

ee iodine in.is method of"Sial, and ournade ai high-'10) oC 109c

)t reprodue-

.rminin¡ free

., COricenlr..10.00 11' or

/ itl.: UUl'4.))O),)

VOL. i', 1982

I\U V ç J j;. .l ....: . .... ;'1 v . V '.. _

ACTIVITY OF POVIDONE.IODINE SOLUTIONS 639

,

J

l

)

!

i

iodine levels of povidone-iodine solutions ofvarious concentrations is necess:iry before: thishypothesis may be adequately tested.

The results of our investi¡;atiol' may haveimportnt applications to clinical medicine.'Manufacturers currently recomniencJ. Cor anti.septic purposes. thai full. strength povidone.io-dine .solutions be applied Cor an unspecifiedperiod of iime. Whether the clinical effcacy ofpovidone-iodine solutions in preventing infee.tions may be enhanced by diluiina siock solu-tions is noi known. 1l is also unclear whether theincrease in free iodine that occurs with dilutionwil increase the incidence or skin irrtation.Funhennore, available iodine is in equilibriumwith Cree iodine: therefore. in the presence ororsanic debris. the concentration of available

iodine may be imponani as a reservoir for freeiodine. In vivo use of dilute povidone-iodine

solutions for antiseptic purposes. however. hasbeen reported by several investigators: theseuses included irrigalion of surgical wounds andpreparlltion for ophthalmic sur¡ery (3, 10. 13).These "reparaiions \/ere described as e8'eciiveand withoui serious side: e8'ecis. Further re.search is necessary. however. before use ofdilute preparations of povidone-iodine may beadvocaied for use on skin and mucous memobranes.

Paticnl.care providers must distinguish be.tween antiseptics and disinfeciants. Ten percentpovidone-iodine soluiion is manufactured forusc as an aniiseptic (i.e.. an antimicrobial agentfor use on skin and mucous membranes). How.ever, in recent years. it has become accepied bymany as a disinfectant (i.e.. an antimicrobialagent for use on inanimale objects). In manyinstiiutions. 10% povidone.iodi/' solution isuse I. liberally for disinfection of blood culturebonle iops, dialysis catheters. bronchoscopu,and other inanimate objeclS. In our study, a

stain or S. Qurtl¿S sUl'ived a i.mln exposure torull-sirenlth 109& povidone-Iodine solution. Thisftnding raises doubts aboui categorical recom.mendaiions Cor use of ¡odophon for disinfectionbased on leveli of available iodine. Manufactur.er have recommended dlMion of iodophorsbefore use as disinfectants (1 2). Hospital person.nel should be awire that cost e8'ICtiveness is noithe only reason Cor this recommendation: thisstud)' demonstrated thai dilulÏni the siock so\u.tion may actually increase its baciericidal activitty.

NOV-0G-iS~5 i0:02

In summary. dilutions of povidone-iodine so-lutions demOnstraLed more rapid bactericidalaction than did full.strength povidone-iodine so-luiions. AlLhough 10% povidone. iodine solu.iions fulfill a useful role in antiseptic praciicetoday, funher chemical and microbiological reosearch is wmanted. If dilute prepar.iions ofpovidone.iodine are found 10 be safe and effca-cious. substantial financial savings as well asimproved aniiseptic care may be realized. Inaddiiion. our results sugsest thai brid exposureof inanimaie objects to undiluted solutions of10% povidone.iodine may be inadequate fordi si nfec tion.

LITERTURE CITDi. A.deMOI, R. L.. L. D. liiclnlcJ, ,lid S. Shiplro. ii Il.

1911. Pscudobacicrcmia aiiributcd 10 eoni¡minaiion o(po-idonc.iodine ..ilh 'si~¡JIl"'O"OS ciporio. ."M. Inicm.Med. 95:32-36.

~. Cr,'eii. D. E., t. Mooy. M. 0, COIIIIOn).~, II. Kollbcb.

K. D, SloUmc;or. ,nd Yo. R. McC.be. 1911. ~euiJobac.Icrcml4 ciused by po-idone.loiJinc ioluiiM coniaminaied"'iih flSl.do,"o"ol cipori~. N. En" 1. "led ~5:6i1-6~3.

3. C,O",pl". 0. O. 1910. Oan,eroui bliadine diipcniin,."led. J. AUII. 11226

~. Foo In4 OniC i\in;ni.iriiioii. i971, O.T.C 10pictJ inii.microbial produm Fed, lIel. l3:1~11. Im-t236.

J. Han.y. S. C. \910. Anii~.piiei ind di~inrietanis. I\nfJ'cides; cctipiraiiliiidei. p. 913. i" A. C. Oilman, L.. S.

Ooodman. ind 11. F. Oilman ied. I. Coodman and Oil.man'i ihe pharmicoio,icil biiii or ihcnpeuliei. 6c~ cd

Maemilin Publiihin. Co.. Inc.. !'Cll York.6. Houiiii. t. T., O. J. A. Cllmo't. C. Riid. and E. J. Sh....

1976. Abicnce or ba~leri31 reiiirancc 10 povidont iodineJ Clin Pithol. 19:":_7".

7. Kin.. T. C.. ind P. .. PrlCI. 1961. An evaluiiion o(

iodophori ii ikin anii.eplici. Suri. O~ne~i. ObS\t1161361-365.

e. Lacry. R. W. 1979, A"lihICI~rill iciivil)' 01 po_idonciodine ioiiard, non.iporini bacieria. J. Appl Bacleriol.

44:44)""9.9. l...b.r,. E. J. t., H. A. Lily, &ld J. P. 1111. 196.

Methodl lor diiinrcciion 01 hindi and opention iiili, 8r.

Med J. Mll-s)6.to. Moccdo. li. E. M. 1971. Prophylui, and treAimenl of

infeclion in eardioihoncic su'ce~ 111111 Biiidinc iolu.

iion. ~. 114I~S. (II TIe P,oclldin" or ihe World Con.llu on Anlllipiis, HP I'bliitinc Co.. Inc.. I'i'" Yor~.

11. Pl'nce, H. N.. w. S. NOlllmiier. R. C, Noriard, iidD. L. PrlnCi. 1918. Oni, ruiiince \Iud;" "',ill ropiul

ini¡ieptici. J. Phirm. Sci. 67:1629-1631.12. Scliiildl, W., ind M. "'1nieo,. 1967. Diier.eni/iodine

iysicmi. Soap Chem. Spec. .Qi61~.13. Shepard, D. O. 1975. Seudine ophihalmic prep and in-

inoculn Iini iuriiry. p. 99. Iii Thi Procecdinii or ih.

World C:onlrlSl on Aniiupiis. HP Publiihin, Co.. Inc..Ntw Yor\.

14. T",ino&i, J.A. 1971. Tile hilo,ini. p. 171-113.111 W. D

HulO (cd.). Inhibiiion and dcsiniciion o( the microbial. cell. Academic Prill. Inc.. LOMon.

r-. i:~

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