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ÏÏTLE
"Development of the nr crnbio] opioal oortrol aspects ofradiation s ter i l izat ion of medical supplies, (part ofa coordinated programme on radiation s ter i ] i^a t ior ofmedical and 'bi^lopical products)
?ÏHiL KEPOKT FOR THE PEFIQD
;''A"
1976-01-01 - 1078-0 3-^!
AUTJCF(s)
Textile Pepearch Institute
>"'""IC EKEKCT AGËMGY
v-
F I N A L R E P O R T
Agency Research Contract No. 1689 / R / RB
Development of the microbiological control aspects of radiation
s ter i l iza t ion of medical products
State Textile Research Ins t i tu te Libérée
664 71 Veverskà BittfâkaCzechoslovakia
Chief scientific investigator: V. CH&KOVit - HOR/KOViCo-authors: P. fiERNtf
D. OLBRICHOVi
Période of investigations: January - "December 1976April 1977 - March 1976
Vorka donc undtr tha Baasaxcn Contract No* 1689/fij/BB nay*ba«n aiaad at following prograai- •valuation of variability in pr«-starili»ation aioreblal
oontanlnatlon of aadical auppliaa rvprtaanitd ly hydropîiillcgaus* swabs and dtMO-apidfcrsuû ipaftêf : - '
- «tap» to oorralat* tat laval of initial «oant* ly usingaub-procasa radiation doa«a and the radiation doaa raquiradfor achiaving aoctptabla laval of pracUcal at«rilitart and
• aiorablological atudita to r«golat« to* production bygianaand the aliJdnation of pathogtnaa from tba «adical
G o n t * a t •• v .re ,
Contants • • • • * • « • • • • • • • • • • * • • • • • • • • • • • • • • • • • • • • • • • • • •
2 «Xf ',2«2*2#3«
2*4»2.5»2*6.
3.1 .
3»2»
3.3.
3.4.
6»7.8.
Stuâls<i problstas •••>••••••«•••«•••••••••••••••
tionEvaluating of th« Influwua of aub-^roctu»6f ganta-radiatioa on dcoontanlnatlon ôfphlllc g a w B abs ••*••»•••••*•••••••••••••••B*t«Talnatioo of ioactlvattoo e w m ••••••••»•Bloch«Bdcal typl»aUon of iaoUttd ata-aln» ••••Statistical • • • • • • • • • • • • • • • • • • • • • • « • • • • •
Fra-atarilisation contamination of-gauaa swabs .....••a*»«..«'*.*...«Suab-atariUBation doaa» and tba dacraasa in -:
noabar of «ontaainataâ products •••••••• . . ••••;;^i«atl«;6f;-'i»i!«r««pgioli»s-: aurriTing tha ir - :radUtlon by aub-atarill»ation doaas ••••••••••Baalttsnea of atraina iaolatad fro» hydropbiUogaust avabs lrradiatad by i î and 9 kd/kg . . . • • •
% "•»••».*•*•.»•••••••••••••••••••.•••*
fapam publiabad OD work dooa uodar *ha ContractCopy of tba pajoar * RadiostarilUatian of damo-•pidaii»al grafta " by •* Cft/lkoré-Horikoiré, X.«uriiakwri, F. Han in pr«aa in Raa. in
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'Jt' •*' »*l ' . i / ' / , V , ' ,'" ' -.. . f « .^-' [..'..
'• . Vv'
' '• '''' '• • ' ' • • < - • • • • ' - • ' * L
'~ - ~ - Ï • ' '- •
to th.
t pr••di««l •applioa udten**, to^prodéci^-;..1^^;<*n-b«;>tnll*
with th* «pplicaUwi of r*di»U»n ;>
*. .^-- : . . - ». . - - • ? ' 'VA. . .
.. .-ji^^i^
«hoio» of th« »ttriU««Uon d«M i» on» of th«A» U to» di»FO»«bl« ••dic. l «sçpU»»
fitnio ••ndlUo»» th« «p«ri«BC« of
tltod» ^ t» th«•ttltod» ^ t» • / th« «th« «* d ^
th» jr « ar ^ *V , • « • Wfhly •oat«^in«t«d «*t«rl«U fir«t #f »U »f
^#f
I à
of
t * « ^ ifc« IMrtii^ of «»• »d itt»t» «Urlllwittwa 4^«»
Daaplta tha eonaidtrabla Mount of aarlg afforta iatha aatabliahaant «T tha art topi» *o* alerobiolocUaX controlant». aafaty aapaat* i f radiation atarilisatiaa yraatiea» th*altaation ramaina aantfarajralal attd as yat n»t aatiafaetaxyaolrad. Xaa raqaixa* atawtar*» ^L such «rittrla aj «M Tarl«aana-tlattal vaalit health aatharlUaa «iffar «tfely /T««|V*»aao«a«n4aUaaa f«i»ol»tad daa to tha Uitlatiira «f UKA
k«k *at tetto^t a sitar aaa*»r.
I» aaaoaaUan idtH radlaUon atwiUaatlona aoabar of alarobiélafUal satnad» waa prapaaaA and
aatailjr im th» aaoraa of last tan yaara. Tollo»-appraaahaa ara Jlx^tly tr lnJlraatlj ralatad ta
• t w i l l V aa»t»l» T - -
af yagaa^aa thaaa hara te ««arantaa ta« «tariliiy« t i l l %tm atarillsa4 *\>at ia ua*4 / U / ,
aorax'aaa MA mÉâdjana <AM^#tosiiUsatlaK aoatttstW* 9 M Bat)M4 lial^f ta aataWiak th«
atartlltatlwi *— aaaar4iD« ta taa «aalraala atarllltr«9«ra« and glraa » r - « i***^*1 «N> Jtffltigr 1» protaatla»that «aà •« ao aoatwla* a«< ka»t ni a« apfva^rlata laral»
- Ta ara-laat* tha afflaiaaar af tfca ^tariUsaUa* «r«aaa«i t la »at aaffiaiamt J«at to htm a talatiTalj «&«• kaa«la4ca
aC tha »ra«atarili«attaa «ao^atuaUaa af pr^aata ta »aatariUta^ aiaaa ooathar baala qmaattaa ariaaa that l*aitatha atariUaaUaa praaaaa, X% ia tha 7aaiata»aa of thata«i mating «laraorgaala— ta « M atarlUsU* m*,***
caaia»a aaataalaatlf I tau prUr ta tha ataxiUaatta»
ÏW taatUc »7 •••»• ofto th* aoat«l of taa atariliaaU»* proaaaa /17,1*,19,2©,*V.
_J
- 3 -
aft-araaaaa dofa» *• *>•••* •* «atl mating «faf araiuata tteaa rtaaia taataslaatad ty al
in,ta» thai* poaolatlatt af produit» aftar irradiai»Ian lay- liât» astbataattaBalls Invar thaa tb« prooaa* atariUs*atU» âcwtt* ' -ïha nfjfjaiift farfafiay at vaciaaa bact*»l«l atrais» ia»>-atad ttm aiMleal pvaâiMta, pra«wUaa awrirgiait and yatfladi«a«|2]r i«ralir«< i » Htm «anafaetwra was' takan oa » ba»aafar a*î«»lifhiB« to» attriliiatioa de«a by QtOOtweta «kdSIDUKaU/22/« *• «alaiOatt tka «ittlsM «aaa Xbê Initialodtmta af •iuraorfattiaas an ptftdoataf tba raaiatanoa atitfolaWt atr«U* aspr«saaâ ia IK10 raloM, and tha raajairad
«T «afatâr «ar« a#rtal4*ra«V ftquaatad areaadttra halpaU a t r t a ^ a* aal««tta« af «aaalUa «aalatamt «utaata,i a yroblaa iaWvaatlag f»r «aav aclaatitic «azkam
ta*t««% tatamlnUg tba imaetlrattam «vrras at atandattvaalaiaAt %* laitsiat raAfttla»* fba «atlxatlaft af
aurtia «•• raiaaaaadad am aaaaiaalaalac a» ra-wttaiia» plattta /U»28»«l/»
Rautt»a «amiral af HM atatilisimf pr#«ta» la inof ^ f^f |aa^ aan^tarf* i«a«
ft ai«m aaabar «f «alls af a atftftftard raalataat
' A •al«al»î» iafatmatla» aft tottraa af aUriUsatlon laay ^ t t i , ^ af dal4|yarata^y jtfttflftataé..awilaal agadyfta»kaaiatani miaraarfanUai» ara iaaattratad in tha asflsemmattttint ia eh«rMt«ristit for ta* *lTan kind af »x»d«at*j andaa tea âmai aat iaflaajwa «f •vrvmûimt aamdltlaM am 1*-araaaaaY dttrtaa* IK nanltJ¥l,% of adLaraaa» «an aa aattmaV•«/31,3a/.
K«vi*ving tho alerobiol«cL««l. attfaodfc toau«ct«d with tfc»«bolt* of th« «UrlUB.-Uon <*>•« and th« «ttrlUty ©ontr»l
^ b i ^ h «t rwxdo* mmt b* aonUaiMt /U,33/« *** M*»*criU«lMd, for w wr«ftli»tte^Jy hi^i nabnr «f•«q>lM t u t t o b t it«t«A in «rd«r to•igniflcwit rtvmtf /349 3V»
1
<1T«B•«•«•17 «ftor tfa* o«B«i*«r«bl« •*! «fftete t» •s
«fl«i«M7 imw»tad n»t bt limit** tgr **•»*!•**,
«bet*elt la, pttttttl «x^angt «ai ytroèu»* »f »tai«*l
«Mtrol •?f It 1» t
prebltm» «•«at«t*â «ithi l i«»i by 1«»1>U« T«Uati««* A écHûtlw* fo«l of
«t*HU«ftti«b pfeOfMMTlt to pro4tt«* stMil» «bjtcts and a»to* A»er««M «t «Iniaat himaanl of • fOMibl* iartetio» offiktlottte* In thi« r«a»«tt th« aiar«Bl«lQflc«l eontrolb«
3 t « d 1 • d
tut wtzfe» two »«*n «1M« «I 4»Ur«tn*tion «f tbt•fftdwwjr of tta* r»U«U«a «t«lll»»*ti»n K * t t w » «MU*°qt i—> r*dl«u«a «od «vftittftUta of * «Mitftbi» Mtbud tcontrol Xà» «ttri l i tj of dLtfOMoU ««dical
foU»«Ug qwtlaaa shall bo atadttd in odar to avalait» »o»»of abova disooaaad yrofelasat
vlfr«fvga>»l«iui e«ata«laatiaf pr«AtMt* Kior toIrradiatl*^ aaâ tlM rtjatsatttatioa tgr Vp*«t
ixtamibn of cwitwiiiwtim jd,«roor^wliM 'in* toolimatle «o—onfcl obangMy
- ilatrlbtttioa of »i«?»t>r«aoi«* *n prodntta *• b«
» door**** iajproportioi i&f tonturtwtoa prodaots idTtor
- réot«tani}« of strnliM itolattd txvm yroduet»r»diat«4 by »^b-pro««sa doa««,
• «tiUrtoM» pt wxrtmtto
- p«aaiU« «rauiaUo* of atarlUly fro« ataUaUcal point ofVlOtt»
2» M • t h • d •
2 . 1 . Sfrukttod n*Wrl«l
fho works ho*t booa e«rrl«(i «at on aapplo* of kgrdxo»»turn* •**•• / 5 • 6 at / talk** roteloxOgr out of tho
prodaotlonal l i » o tgr *h» nuMMKf*etur«r for t^« p«xi«te «f « M
200 pl*e«e of*t to» «id of
mk» «in*SU»10
«roxy l w t
Ibe 9or«Hit«s>lU«atita touat» ofu m i i i l » iè# 0TW3T t i m t grfff'»sr 10 »aqpl««, «W.X* th»4tbtr groupe «•?• dtt«mla«d ÎN» «t^gr th« iAf luone* ofpcooo* 4p**« of
2«2«
th» y«lcroorganlas» th« s«Bplo* we»« rimiod for 30 «in in 300 a lof sSrauMn't b*ff«r /$h*t92/* ttw& MCOMAXT-, tho xi*»*»100 tU«« dllttttd «Ad forth** CLUltr«t«4 through a«mbr«o
• C*tch prodvtt srxiOR / 90 m «1*0» anroraco po^ro « 1 M<•»/• f l l t«f« wort iMtibatod 00 platoo with »lood ««or / 0 » i d
CM 55 wd 5 » of 4«fiorln*to4 «teop blood/ tê* 2 d«or» «t «of 25 *0 «ad«r MVOW« m& « M n t t i otmoijMo—*
ool fy aorphologr ond ooKVtntloool «loroooopgr of tr«M*•t«iaod ptropafttUoM woro aarlo»
•t-
•t«ltt»ttn« the rooalt»,,tbt «Mv»ptl»ft us» udo:tfeat «M «iartttil eolevjr mi «reatajt toy 4irl«ioa *f « M ««11.
2.3* of tfa» lnttaoneo «f
mte
I* »t«4y th» t«fi*iiMar iT dtff or*at 4OM* at p a|
1b* MMpl«« of gI P -
dot* If I , 5» T, • , 11» 1$ m i IS W/kC / 0,1 - 1,5 a n * A& *° . ID14
SÎMHM «qpuîatata nw* T«p««toê «irwjr «Mk «vtr «b*fM 7§«r a» will M III» f t i—t i— of
«tlom tfiMÉp «ff «l«r*«fc«itiiMi «« proiMt» a«£lA«t«d atfro» proftxtloa
• fI tb* I» ttvt fl«eo«
Aftery e j 10 Ml of B»trl«at
trot* / W«f lM«rt «xtrMt 1009 «1, y^tem 0,2*, M81 2% .
i «h» atàltif* oX tko Mtri|i«Ml M«1«>I MB »t*f»»N< .ill « 1 «*••!• 1, fl— b«Mli. «• oomtrol tlM tookaidtM t f ororlaylB<
ty tin» nutritional atdlua and » poaalbl» aeci&antal eontaainafe»Ion two additional «toril* tosVtuboa / without any gaus* avaaa /in arory groqp wor* f i l lod tgr tt» » * M JMdlai and eultlT«t«4under tia* mm» condition* M th» t«»V-tob«» i»ltt
14 âigr* of laetiba-tlwa 1b* fnmth oflan* wu obs*rr«A. fh« sarrlvinc strain* twmk Isolated o»plat»» «lth flat» Count agar /Oxol* CM J2i/ to ba idantlfladblocbMileallar and «ralttâttà £ro* «at poiat of radiation r«ai»V>aaca»
tit <i2*4»
rtalataxut* of baetcrlal strain laoSAtad fro» hydro-phtlia ipuair •»«*• aftar irradtatloA -»aa araltaatad ty .Aationaia»atlon of tnair inaotlYatlam «ttrvwa «aft ttal«ol«tln« tha laaattv»aUoft
«»ra aatlawtad for tao1
aovreaat
Jrradiaiar «rp» J-MOtt, «at* LW., aatlvlt» 4»fa • 30 .at Ifta b«eUBia« t f
atraiM Irara la«uaaU4 ait natrloataf«r/0Da»14 « V for 4« a aft* ayot^fonUc atralaa fa» f «aya** ta» a»Ua«l W«»«raV»a. i f tor laMbataaa a najgyaUn im0,0? M paaayaaf fcafft* wa» pr«F—* M i » 4 < n M ta •%arilt
taa^. Tb« aoprtx. «MWMtraUoa ma ^ ^ « « U a or
' '- •--..'"
in 1 «1 of •uap«n»ioii, Ih« t«»t»d
; - 1,5
were furtb.tr k«pt\ : ^ ; y : - - ; ; \ - : ' • ; , • • J - . ; . • • : • £ • : • • •• •' • / ; • . / . ; ;
«f t««it«â stridap v«r« irradlettd tqr . ' ; -
«nd lir^to
main
in 0,05 M sSr«M«n'« bufforof .:<^'1«1M^ ^ ^ l ^ e i
CM
' '•' - ;
,2/. Th» «Munt» ©f 0,1 «1 ";>: -.«n I»!»!»» with Pla-t« i
«t 35*C for 3«t roe»
^ ^ ^
^ ; , J ^ ; for«^ e ^ â ^ . Svapëtwiona or b^^
plp«tt«d in 0,1 «1 «Mont» into V
•ftor lrr«4i«tio» th« ••a>l»« wr« «u«p«4«d in 0,05 Mboffor /^U7,i!/ *nd ho«0f#niM4. ?ortt " "
^ ^ ^ ^ ^ , , « i f ^ | # d ; ^ | < w i r .
f^ aritb»*ti»M of
fratiloidi «f iorriyiait Éi«rooygkr>l»•>/ 1L/T^ /w>* plotted -
r ti«b»
^ ^ ^ T ^ ^ ^ ^ ^ ^ ^ ^ ^ J ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ * * ^ ^ T ^ W ^ w ^ p ' ? " T ^ ™ - t l | P ^ ' a r T V ^ ^ ^ P ^ W H ^ ( | ^ P T I ^ ^ I B ; ^ V ^ ^ ^ ^ V P ^ r i ^ ^ ^ ' - J I P ^ - ' '*- * " - , ' ' ^ ' " f i ' r i > t f ' *
of
i«oX«t*d ^ a f^ tgr «ub-
Or»«i«LD«d
«e
f««*nt«U«a of l prodacUo»,
r»l—»t ^aytlmt» Iwwly»!^ »r«èwtU«a »»l—»t ^aytlmt» Iwwly»!^ »r«èwtU«a »f j y » *fP—« «f »>iwyT>1 f t w t f<y—mUtlMff <!!>—••, «—alt
/ f^j lp:^:]^^
W /37/» 1«D» / W , » U » /31/ « ^ 1 » ^ ^ : "for T«a»«qr *f tU»tagrlM««el ««d «KroMMi740/
( :
*':'•••'••• "'•il - ' ' . I * * \ T . i . " ' )
'/'-^^up^/Of!/«|p^i««iii;^è3^pc^«^4^ow;'1in:^ç«^i-^^^
prowdor«i a - v»ltt«» rtt«itt«d i* tkt »*»iitd »onto i»«r« di»-trlbat«a in k - i l u i In tmal i , 1b* *W«l«t* fr«q««Bey of
v ;;. MM$ï^k • • î##-
of iat»rr«l
W^•^i^î^l^v^
I
* ••*proridaS \\m condition 1* folfiliad ap* > 5•••» k»
! K X > 7V^ /k-V tha typothaal* for Toiaaoadi»tributtoaia nmbar of •lereorganlp^a o» t«si>-p&«as» ofhyâropMUc g«os« aw«b« at << Mnalflewte* l«r»i i» roj«et*4*7«lm Xj /k-V la tb« critic»! v»la« of th« Polawn 41»trlbat-ioa for tlM •< plinifleaK* 1«*«1 «od k - 1 dtfroo» of «ro*o»»»It lo xxfd in tablo / 4V *h»r« ^ - q / 200»
$h« of th» paranatr A that ia «qoal to«oaa valut of thia fliatritmtian i * jivoa tgr:
«har* a j ia th« eantta of i - «laaa intcrral*
2.6,2. , fh» taat to v»rify th» fagrpothaaia forprobaMlitr alatrlbtttto* I A noaaar of «iaroorganiaaa on hydra»pallia ta?*a awaba «aa aamioi ant ia gat a aanaral laforaatlo*ty plottiuf tho data o» laft-aental papar.
danait^ of th» l o f aoraal diatribtttlaai la gLvom ayfonwlat
/la x "
far x >O
t M
• •' X'
of
2*6 «3«toditd
to
-. V
to FOpFtcoiitAto wtyj T flJTWT iwiiiÉPtiftfi 1jhj> t*>nfkmt T*V>1CT^ fsnoKtho Boiaal d io t^buUon i l t h p^»
lagr B«rt^ott*« toot•. 'iftor ' thO: dlfforoneo* i o aaan- yoloi», forindividual iwnt*»» ^ro; -;«volttâto*- tsgr •• di«pf jolon onoljoi», of, tho•iapXo 4Loporaioa aulyoi* : wo« ' e«tried out
Vc/, for
DM* of e«l«ul«tioiis aoo«rdliic ' to^^^-'';;'''-^r;;:,^:-r;.."-'-;-::";:\::-^..j.
of • • « *•!»»•• i« rt J»oUd i f ;
of ïl»V»rH5Mâ«epr 4l»til]buUo^
diff•!««»•' trttwi I A U McounU «ko fco^twlij i ndoal tn«te ;wM;«T«lu*tod 'kar; Co^f^
thl» to«t k ditporoloM caa )Mi o f :
l
o ':•• x
; hi-
_J
- 14 -
Table 1 : Characteristics for dispersion analysis
Month
1
••••i
*• , '•
* \ *
a=12
Value y i ;j
y i l « • • • ^ i i ^
y a l ' # # *» r an a
£ y?- -
ITumber oivalues
n l
*•••n i
•••na
N =
S 3H53
h.
••**
•••Y a .
Ï =• •
" i Y i -
Aeaa valu<
••••y i .
#•»
y . . =
n i
*ln i••••
*•
na
Y2
K = - " • =K
iliwïtere Y _ v n.
i i
i .
- 15 -
Table 2 : Dispersion analysis of simple classification
S
Saarce ofvariability
Betweenclasses
Error
Total
-Square sum
asA =L n i / y i—- / 2
a n-
Se= I I /yi ry ± /2
e i=l j=l 1J lm
i=l T=l
Number ofdegrees offfeeûoa
fA= a - 1
f e=r N - a
N-1
Meansquare
MSe= |s-=s2e
The teat criterion is:
C =/s?/
231 +
+ sn
where s| is the estimation of i- choice dispersion, i=l,2, ..*, k. baseâ on f degrees of freedom /f= 10-1 = 9/«The critical values C are noted in table /4V» The hypothesisfor dispersion equality is rejected if C C /k,f/.
- 16 -
. V
2.4.6» Confidence intervals were computed to evaluatethe fraction of test-pieces of hydrophilic gauze swabs thoseremained contaminated by microorganisms after exposure tosub-process doses of gamma radiation.
Confidence intervals were calculated for the fraction Or/for individual weeks and for various values of the dose D/ rijj was the mraber cf teat-pieces on those bacteria survivedthe dose of radiation D and OQ the number of control unir-radiated test pieces / •
fir,The confidence interval for S ^ has a form
where/ f
l ' V+nD
f l "
/ f /f /fl'V
F /f l f f2/ are the critical values of Fischer-
Snedecor's distribution with ^ and f degrees of freedomthose are noted in table /4V.
was
^^ $fipfi$ ; ^ ; of in«ctiT«tlon eurr» -; ; _
^ -
ofvlth «n
• .' *"• 'j '.- '" '" V '
• . ; « ? , . - . z ',*'
' - • V - - - , . , • \
- log»rltbffllc txiiMfOTMI
}}
r*lntiott / 1 / ' « f«t », ^ :
- 18 -
m m m
ni
fm
A ' (il, lnND.N Q = e
m
'; Carrelation between theoretical aad etapirical datain the relation. /2/ is expressed fcy coeficient of correlationr " between. D and In T>n,It holds:
ni m ni
r«-î=2
r^Cr) are «cfc criticalIf Irl > r^lT) , (rtf= m - 2,values of coeficient of correlation that is noted in table /AX/*Then the dependence In N D na D can be considered forstatistically significant.
XuBâtlT«tl«a curv« oxprtfosod ty «qtuitioa /3t/ «haraetoristdtoo roairtw&eo of •ioroorfanisa* foi* M oaUod * •*•* * **dioVion £»••*; Hi* nuafeor' of surviving xdoroorjgnnioa* i f «ftèii
*t dts«»n«ér tt *tr» than «ftrrti^oadiac to etlcalationsto «qiuitioa /3/« Sorti dtpftndcntt* ««a. b« s*r« proptrl?ty follotriac
Stt* «oiwtaDt « «barMUrtM» ooor«« of i»««tiT«tion c o mia r*fi»a of «i«U r*dl«tion dosait fi* SstjbiatioM Of pprwwtt»itt a «ad 9" wtrt coaput«d <*o« «quAtioa gkirott fo* £, »#
r a» d««<xibà4 abor», feat tMÙtd ^ th«/ 1^ v*» taktm M
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- 1» th« doi« of radtrtio»,- ttttibor of «i«roorf«dBM iurritint «ho
Jl# - atwiM* of aievoorf*Bi«M prior to
. ' ' • * • ; • " • . , ' ' . ' • . ' " . ' * " " • • ' • ' ' • " • '
' • - ' ' * • • • • % ' . . ; . • ' • ' - • ' • ' ' * ' . ' ' - . ' y ' - ' = - " - . - • • ' • ; • : - ^ ' - - "
v ' ' 1b* • i>r«^«t«riii|t«tlon b*çt«rt«i - couata. «a ttarârephiliegaos« «wab» ttJten froa production »t r«n4<» W«M
of on» y««p. Ih«
for Foi*»ea Alctributlèa of viex-oprganlans
«qoal for i a i dati iA^^ t
^ of th« #tudi«* yw.^^^^^
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»t«U«Uc«ll3r «i«»iflc«nt «t 1 * for Oet«b«r, »^r«b«r, Jiaxoarj,April, M*y »n4 July. On b*-4i« »f th«»t rwult» »• had tc r«J«*tto* hgrp«to«»i« f«r >*i»»«n 41«triWUo« »f •ioxo«r««ni»«» on
"'\. 3taf»r*«tton «rwf of hjrpothwi» for l»t»oi«»l el . triUn w» ••*• *(f ^l«tUn« t»« «xpfrtawiUl **t« »f *t«7 »o«th
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- 21 -
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s^
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cs
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ft<a;
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ooo
on
rH
oK
ool - <i H
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O
a
2400
enoen
H
CM
OC
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OO ili><M
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330
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O
S
1027
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co
6990
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Oen
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Ci nCO
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920
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CM
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CO
CM
CM
ol - (
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082
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1492
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5120
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5040
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- 22 -
Table 4 : Calculations for test of goodness of fit forPoisson probability distribution, of microor-ganisms on one product
t
Month
OctoberNovemberDecemberJanuaryFebruary,MarchApril
May .'•Jane .'JulyAugustSeptember
n
4940
20
38
38405040
40
5030
30
k
6
52
546555544
46,2528,505,65
51,588,39
11,6660,1517,8013,2086,56
9,73,71
X2A-1/
1 1 , 1 *
9,49 *3,84 *9,49 *7,81 *
11,1 *9,49 *9,49 *9,49 *9,49 *7,81 *7,81
T?/K-l/
15,1 * *13,3 * *6,63
13,3 * *11,3'15,113,3 * *13,3 * *13,313,3 * *11,311,3
4 , 1
5 , 1
2 , 7
4 , 3
2 , 6
4 ,91,9
3,91,92 , 0
6,35,7
- deviation of empirical probability distribution
differs from the theoretical one at 3% significance
level
* - deviation différa at 1% significance level
* 'V
• s f
,*g -
. -, *, ..: - - - ' * '"* >; *
ft
\>
tor• cwaomlation of point* to tb« ste^gjbt l in t 1» oxo«* « ^ « I ^ B J ^ - :by Montb^ia wWcb thé y bacttrial counU on t t i t -
1 ' :.'-/-
'•V-:*ia}fwa»- ; pf and «tadadard/È/ liMit* «r 95* cbnfid«nc«
lAt«j^*X for pa^aattilcal^ ^ «nd i t» «fitiMiiion7 ,«^,,:,'•ir« coApdtvd* A t oonftdtne* intcrral 1» for aa«ll TCLLOM of -v
' of tbl» di«tribution» !»•* for iwtr«^ nv»btr ofon «iaiHLt prodoet* of bydrophilic g«vw« iiwabii,
data «art *l^«n in Tigurt ï t in «td.eh
i ^ i ^ E i ; i s ^ ^ ft^r^%ië^ t î à i | Â à M ^ è ^ i ^ Ê :pblliewer*;tf«teâ,;'oa
^ out of the prod««in •èriabilitar i ^ i ^ indt^dual
in
by Cty * o n
oo» ««nth not found W b« «Uti^Ucklly • igo i f i cwt ,
of »••« T « 1 « « for
- 24 -
Table 5 : Estimations of mean value EX and standard deviationIfiX, lower D and upper H. limits of 95S? confidenceinterval for parametrical function e^ and itsestimation
f
Month
October
November- .
December
January
February
Week
1
2
345
1
2
34
12
1
234
12
34
AEX
104,39,37,
6,39,
24,104,
80 ,10 ,
1652
2154130
9
993
11
41270292
15
4282
20
68
,60
,01
,36,17,34,40
,36,34,71,67
98,23 ,40,
4 ,76,
16,57,
102,15
1360
388629
4
793
2 1
X
02
77231525
147785
,25
,16,37
,58,06
,76,49
,30,99,50,91
13
43,2 2 ,
13 ,3 ,7,
13,63,24 ,
3
317
1866
11
6
431
2
10
52169930
245036
,04
, 01
,53
,66,90,92,13
,50,42,34,44
H
134,4250,1147,711,1643,78
31,33132,7
99,8012,33
12,5465,73
240,7545,7339,4211,74
12,0811,925,44
13,76
76,33,2 5 ,
5,17 ,
2 0
9149
6
633
211
172 1
8
762
5
ty12
60
079488
,37, 80
,31,13
,15,95
,98,77,68,48
,37,38,70,78
Table 5 continues :
- 25 -
t
Month
Karch
April
.
Mgy
June
July
Week.
12
34
12
345
12
34
12
34
12
345
46,065,034,134,97
14,845,165,75
10,3921,92
31,0914,7537,558,53
36,5026,4913,6423,03
T9,7723,055,609,163,52
Vox
7,716,023,975,39
37,4511,885,02
16,7319,47
18,938,50
28,371,88
43,7619,1011,1618,84
26,2622,16
3,253,252,79
D
1,861,651,663,44
1,990,832,532,449,49
17,768,71
18,527,12
11,9013,536,33
10,37
60,229,413,066,751,68
H
7,566,34
10,646,11
15,025,537,42
12,3028,27
39,6818,7548,44
9,74
91,8931,1317,629,74
95,3229,807,65
11,054,57
3,8
3,232,974,59
5,462,144,335,48
16,39
26,5412,7829,95
8,33
23,3821,4910,5617,82
75,7616,744,848,632,77
- 26 -
Table 5 continues :
fMonth
August
Septemt
Week
1
23
er I23
EX
62,18111,9269,98
66,0842,4095,33
A
\fiaf
24,2035,6917,02
27,9722,8497,85
D
44,2985,3457,27
45,5026,0136,25
H
151,60133,2280,73
81,3655,51
122,04
57,95106,6368,00
60,8537,3266,51
- 27 -
t
Table 6 : Results of testing homogenity in variability ofpre-sterilization contamination for individualmonths and dispersion analysis
Month
OctoberNovemberDecember
JanuaryFebruary
March April
MayJuneJo""
A . tS f.K member
Values n^
494020
383840
50404050
30
30
Mean v«y^
3,153,312,673,38
1,741,30
1,68
2,842,86
2,39
4,324,05
1,40
1,68
1,642,18
1,03
0,641,38
0,64
0,641,650,16
0,61
Total: sA = 347,71Se = 524,05ST = 871,75
fe « 453MSA = 31,61MSe * 1,16
F/fA,fe/ = &/11.453/ =1,792* < - 0,05
reap.
e - . - v . ' - . — ' - 2,253** < = 0,01
- significance at 5 %, resp. 1 % level
- 28 -
Table 7 î Results of Cochran's tes t of dispersion homogenity
Month
October
November
December
January
February
Week
12345
1234
1
2
1234
1234
Mean 'X
4,33233,51453,22151,78122,8837
3,01394,51963,89811,8128
1,81643,5249
5,35652,87753,07632,1380
2,22061,85360,99421,9504
Sstimat.ofdispersion
40,63210,31230,77990,30681,5674
0,36260,26530,97271,1115
0,99250,8532
0,03161,68070,67250,2061
0,47500,76210,63531,4634
Test cri terion
C = 0,4356
C o > 0 5 / 5 ,9 / = 0,4241 *
C O,Ol / 5 > 9 / = °»4854
C = 0,4098
C 0 > 0 5 /4 ,9 / = 0,5017C o | o l / 4 , 9 / = 0,5702
C = 0,6487CO ) O 5 /2 ,9/ = 0,8010Co',Ol /2 '9/ = °'a574
C = 0,6487
C O f 0 5 /4 ,9 / = 0,5017 *
^ C 0 , 0 1 / 4 » 9 / = °'57C2 * *
C = 0,4387C0 0 5 / 4 ' 9 / = °»5 0 1 7
Co |ol/4,9/ = 0,5702
Table ? continues :
- 2 9 "
t
atonth
March
Apri l
May,.
Jan»
July
Week
12
34
12
34
ITS1234
1234
12345
Mean :X
1,32121,17311,09411,5238
1,69820,76241,46621,70112,7966
3,27882,54793,39962,1203
3,15183,06772,35672,8804
4,32762,81821,57562,15591,0218
stimat.ofiispersioi
40,96230,88720,65280,1608
1,99771,75610,56591,27940,5819
0,31570,28710,45190,0476
0,89000,41840,51240,5126
0,10300,64950,29070,11870,4846
C =
cn
c =co,co,
c =
co,co,
c =
c0
co
C =
co
CO
Test
= 0,361305/4,9/
0 1 / 4 » 9 /
•' 0,3232
O5 / 5 » 9 /
Ol/5.9/
- 0,4100
O5 / 4 .9 /
O1/4 .9 /
- 0,3817
,05 / 4 ' 9 /
,oi /4 '9/
* 0,3945
> O 5/5.9/
,01/5.9/
criterionl
» 0,5017= 0,5702
* 0,4241
= 0,4854
= 0,5017
= 0,5702
= 0,5017
= 0,5702
= 0,4241
« 0,4854
- 30 -
Table 7 continuea :
Month
August
Septembei
Week
123
123
Me n -
4,05954,66944,2194
4,10833,61974,1974
Sstimat.ofiispersion
3 i
0,14100,0969 •0,0576
0,16490,25490,7197
Test cr i ter ion
C = 0,4771C0 O 5 / 3 ' 9 / = ° ' 6 1 5 7
C o' ,Ol / 3 ' 9 / = °> 6 9 1 2
C =•• 0 , 6 3 1 6C0 O5 / 3 ' 9 / = °tôl67*c o!oi / 3 ' 9 / * °«6912
resp - significance at 5 %t respectively 1 % level
- 31 -
Table 8 : Dispersion analysis and homogenity in mean values
for individual weeks within one month
Month
OctoberNovemberDecemberJanuaryFebruaryMarchA p a - i l '••
Majp :< '
June1
Ju3#AugustSeptembei
8,69813,79414,59419,19813,9947,6895,3373,6961,275
16,3620,9860,967
FA
431
33343342
2
MSe
0,6900,64940,91830,40140,09260,26800,78860,37060,55330,35060,06550,3566
•p
*e
443618
343436453636452727
N
4940
20
383840504040503030
F
12,6021,2415,8947,83
151,1228,69
6,779,982,30
46,6715,072,71
P0,05
2,602,924,142,9222,922,922,612,922,922,603,353,35
FO,01
4,024,518,284,514,514,513,834,514,514,025,495,49
Sign*
* ** ** ** ** *
* %* ** *X
* *
X
* - significance at 5 % sign, level
* * - " at 1 % sign, level
x - " at 10 % sign, level
sce•H
U- 3 2 -
ce
ICD
fcO
•ri
<H
u•Hto
COCOQ>
11
Gr+ rods
•Gr+ spores
Cocci
i
•
i
*om
O ra• u
Month
CM
o>
c - l
OS
18,8
O SCM
28,5
47,4
P
154
h
O4=
S
0,85
1-4
1
26,9
40,2
29,1
117
u
I
1
1
1
1
1
1
29,4
ITS
17,6
OS
52,9
Htr\
December
i
i
00
OS
CM
CM
HH
H• < * •
29,6
Os
27,3
VOrn
CM
H
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1
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t -
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- ,
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VOCM
34,0
VO
32,1
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106
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7,37
H
1 0
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1
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24,3
CM
34,7
m
27,4
vOCM
ITS
os
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4,30
i
35,6
32,2
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27,8
115
April
Hr-i
«0
2,0
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t
24,0
45,9
VO
24,0
s
146
I
.n
3,47
UN
0,87
•H
22,2
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m
31,3
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40,0
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144
June
«
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1
1
M
C VCM .
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44,3
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2,8
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UNi - l
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16,1
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49,6
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143
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34,0
om
19,7
OsCM
44,S
vOvo
147
tr
1,61
ITSCM
5,61
&
0,64
o
25,3
393
29,4
456
37,5
285
1 553
oH
- 33 -
Figure 1 : Pre-sterilization counts of microorganisms ontjydrophilic gauze swabs over the période fromOctober 1975 to September 1976
oar^ v : »iv .HH;1V'cn^f i ie i ir i i that rtault» of inditidaal. w*«k»;
d^f*r aijg^ficant^ in a --:.. diff*r aijpiificant^ ^ al l aonth« *T*r ^« p*riod* of on* • - ''C 3r*af at 5ft ai^odfieano» l«ir«lf r«ap«ctiv*ly at 10% in Jon* and
Sapt««b4»^ ^ f diffat*iW*a can b* «a* ^ la fi'gttr* 1.
ogieal
in
of •lcroorganiaa»t>T«r on» y«ar ar««ra«'.;r#ppi*tnt*d>'% cocci / 24 »
atrobic apor«-forai«« ro4« / 16,1 -
odntaainatfd produatf
oo»-
pariod* of on* 7*ar. Th* atti*«*ri«*d r*»»!** of individual-; ' '»ntto.,.iw ^t*^;>iattp«j^^-^:;^^*T^uati<>a_ of qàrt*r* and th* .; , total y«ar in tabl* 11» C*nfid*nc* MaA a lnt*rvala w»r* coiapuV
', ;;'•d;.for:\fi'a«Uo^; »^^-;i*l*^ i»f:^.'Mb^:>'*^r^t^^«iBf.«' }--.
' ' b * * « ^ ^' faOM awab* ia illoatratad lç n«ut« 2 and 3 for • • • i r i t * d • / - •
'' ' ' ^ ' ^ i w - ^ ; ^ ^ i y t * j ^ " V .t. .'; •;•/•'.' ;'
: ; . , ' • „ • • * - - - ' . . i: - , . * • : ; • • - • ' * • • ; • • . • • • ' -
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- 35 -
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- 37 -
Figure 2 : Inactivation effect of increased sub-sterilizationdoses on the microorganisms on gauze swabs:summary of results of the 4th quarter 1975 anathe 1st quarter 1976
NdNo ,
0,8 •
0.6 -
0,4
02
IV. 1975
}] x I01 3 5 7 9 11 13 15 D(kJ/kg)
NdNo
1
0,8
0,6 Ï
0,4
Ï ÏI. 1976
J l01 3 5 7 9 11 13 15 D(kJ/kg)
- 38 -
Figure 3 Î Inactivation effect of increased sub-sterilizationdoses on the microorganisms on gauze swabs:summary of résulta of the 2nd and 3rd quarter 1976
No 1
0,8
0,6
0,4 1
0,2 1
} II. 1976
I I I 1 I I I I
01 3 5 7 9 11 13 15
NdNo
1
0,8 '
0,6 "
0,4 "
0,2 •
?? ç
i. 111.1976
Û01 3 5 7 9 11 13 15
- 3 9 -
Figure 4 : Inactivation effect of increased sub-sterilisationdoses on the microorganisms on gauze swabs:summary of results of the total year
Nd .No
0,8-
0,6
0,4 H
0,2-
Î Ï
] X I I0 1 3 5 7 9 11 13 15
D(kJ/kg)
V " ' ' • ' "
' : -;--i--.iTO»;çb.t iB«d;'>j«iaiat«, i v**^#^«*vt&«t th»
••rt.»T« \\A- a « n »vf l»>>nvhtt*L<<itty T'f th< '^•«ttf l i i <yf b o a i t i v a <rowtb: roaolta to Xbm;ii}iiaSi^:-wiii>M^of:- aaaploa ^ tcatèd i t plotttd «gaiaat
' dépëndoitca. of \fraetiott of;wa»:
of
1,3t«st-pftècë ox*
3.3» froiittlon,;of
doa« w«« M«d«da«^abo««^ .;'t^^:«ai V^Uott t«cbniqoa' bf';tb»' aftbodastd» hovéTcr, no 'fow¥l «ft«r irr»dUtioa *3
« tdf^ *•»» >aloi -ai:'c»tart-.2t,4é6«;::;.
>ttrylvinjt y » lrr»dl«tloa;
Str«lo» i»>l«t*â M tMt-pi«««« Of hydrophili»«««be tbCNM «tr« Irradiated tor «ob*#t«rilis*ttoaldmtifltd éa fe«i« of aorpbologiQAl «ad Pioeb«*i«alf*aa ttM total aawWr 1?T l*ol*tad «tvaiMy M «*r« Qraa po«ltiv«
rod» of twm Ba«lllMt 4» ctralna o»long#d t#*o«d m& X atealn of Qram o*gativ« r*i «lfavut roda of tb« typo
tn fabls 12 à ttateary of id«Atifltd atrala* iaolatadfro« CMS* Mraba irr*4l»t^ If %t % ? and 9 «*/kg /0,3-0,9 «rad/
- 41 -
Table 12 : Summary of strains isolated on irradiated gauze
swabs
DosekJ/kg
3
5-
?
9
9
lumber ofisol.strains
21111
47223
614411522863142121
13131
Total-
6
84
28
8
1
Identification
Bacillus subtilisBacillus coagulans
Micrococcus luteusMicrocoecus roseusStaphvlococcus saprophyticus
Bacillus cereuaBacillus polymyxaBacillus alveiBacillus coagalausBacillus megateriumBscillus subtilisMicrococcus luteusMicrococcus variansMicrococcus roseusMicrococcus sedentariusMicrococcus agilisMicrococcus nishinomiyaensisMicrocoecus lylaeStaphylococcus epidermidisStaphylococcus saprophyticus
Bacillus subtilisBacillus cereusBacillus raegaterium
Micrococcus kristinaeMicrococcus luteusMicrococcus roseusMicrococcus agilisStaphylococcus saprophyticusStaphylococcus epidermidis
Bacillus coagulansBacillus subtilisI^cillus pumilusBacillus cereus
Serratia macerans
f '
la flyan* Tm« «aat laaUU* atiNrtJat a«oni •••«I aalonfat 1» Mlara-* I M N I l i t w / U / wt MltTMiiew m i w / T «train» / . As U .. tfc* «*WM B«dllto » ftw afcaalataly fraqaant tyna «M »•• / H »tr«l» / , l ^ t h » K ••bUll» / 1* «U*l«« / , 1./ 5 •twln»/ «>d ». «Mtte^iw/ 4 «tr«l»« / • « f
U tt* *»»» » M/l^ »— —frtT«d by f atraias taaaa *•?•• • » . »«bUU», • • ««rai», » . a a t a l — » » .
—rr»U* • • « t r i a . '. ; '-'! ';•' SS''•'' /:"-.^.^:. >-; "' / .<'/•''
IMM 7 M i I ,7 «< 0.» lr»V «t*
taX In aa4 aaltor—<rtai phaaphaf
;; HM MmUUM «HfltiMt f |lTM «tiiMN iMT « VU?>»laU»» #»twaaa tna éaae and naatar »f
- a% WAlaa
. ï»a aalMla—a far >otH «aattana / V and / V la ik' a i latralna v«qr alaar. Iha al«OflaaM« af vaaolta la alaaaat
af tka »atk «a«ala far naolan a»aas ara aUllar aa 1t.»f 1ka aanatant k. ï :;ÏU'"'"':":-.
.;/-. .; Tba aoMiant $ aaaraatvHMa raalaUaaa af tka aiadlat^W ^n^^^nna^na a a ^ p t'v^^a^aja^aj^i^^a^ 4n> ^p kfa>^vv ' • S^ni nT' ^a^^^pin^BH^ j^ av^a^a^fla^y^p^pm^^^^ _ v^ajup t a ^ ^ r a^Baa^p^paj^^pvvaj
ffea aetaal raalataaaa waa taflaanatt élattMtly ay aa^rlramantal
•wditieo», dri«t «ttlt«r««M it it
i l f«r tiM ruiitMH
UU M « 1 4 wd
rtlw, B. M*UU« « d ». M«*t«ri«B. B/ ««viral «trai*» • di»tU-•lM«id«r «y»««r«d «a lKMUf «U«tt «anrtft «tUr ite
'" «KMJ illu«tr«Ud
»« »f 4
«id
wd B » tbwi w m i a
f«r
13 «od U .wd <• AU
th« for
Table 13 : Data and constants of inactivation curves:Bacillus megaterium and Bacillus subtilis
c
dose
o
train
kJ/kg
0
3
'e
9
12
15
B. megat-^r4 urn 107phosphate
buf ~er
1
1 ,39 .10" 2
2,28 .10" 4
8,50 .10"°
1 ,09 . ! 0 ~ 9
2,13 . 10" '
drisd
1
1,89 .10"1
6,58 .10~2
1,63 .10""
2,02 . lO" '
5,04 .10"'
B. sub t i l i e 67phosphate
buffer
1
3,!0 .10"3
3,10 .10" '
9,5C .-,G~6
8,79 ;io~12
dried
1I
8,54 . 10 ' -
4,41 .10"-
3,75 .1C-"J
?,43 .1C"1
1,68 , I0" 5
-'
9•o
£>
fiM
C
r-iIIo
a
c
N o
k
r
sign.
n
k
r
sign.
m
D10( kJ/kg;
7,03 . 1 0 i 2
1,7158
0,9948
++
3,4.5
1,7158
0,9948
6
0,70
7,55 . Î 0 8
0,6142
0,9738
++
1 ,66
0,6142
0,9738
• 6
0,21
2,51 .101 2
1 ,6794
0,9991
++
2,81
1,6794
0,9991
-••+
6
0,74
- ; rj~ , ^ "
0,6301
0,9901
+ +
C,63O1
0,9901
-f+
6
0,28
- 45 -
Figure 5 t Inactivation curves of Bacillus negaterium andBacillus subtilis
Table Î4 : Data and constants of inactivation curves:îîicrococcus luteus and îîicrocoecas roseus
Strain
dose
o
-a
Pi
o»n
•as
i
s
II
O
ssc
1—1
kJ/kg
0
•1
2
3
4
5
• 6
k
r
sign*
n
k
r
sign.
m
D10(kJ/kg)
y . lu teusphosphate
buffer
7
2
6
2
4
2
3
0
1
3
0
1
1
,56 . ' O " 2
,37 .10"4
,03 .10"f '
,45 .10~7
,90 .10"S
—
,64 .1C1C
,9227
,9959+ +
,29
,9227
,9959
+ +
6
,11
i
3
1
2
6
4
1
0
0
1
0
115
dried
1
,10 .10"''
,29 • ' 0
,70 .10"-
,01 .10"3
,30 .10~4
,03 . 10" '
,06 . !0 / -
,4547
,9754++
, 3 1 •
,4547
,9754
+ +
7
0,83
M. roreus
phosphatebuffer
1
7,18 .10"'
8,57 .10"-
8,20 -10"J
2,07 .10"°
2,87 .10"7
—
1,13 .109
3,2871
0,9461+ +
9,02
3,2871
0,9461
+ +
6
0,50 \
104
d r i e d
2,
i
3,
9
1
C-
7
2
?
2
0
0
1
62 .TO"1
*- -^ • i ' j
61 ,1C "
48 .1C"4
59 .10"-'
, ^ y • •• !
,60 . !0 S
, 1 526
,9829+ +
,39
,1526
,9329
+ +
7
,71
- 4 7 -
Figure 6 : Inactivation curves of Micrococcus. luteus 115and Micrococcu3 roseus 104
v.
t * 0 m •a
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ofI t bj« b«»n «t»t*d that for ao<»t«adn«t«û with Mr«Utt«»« mitrovefaXmm m i»
lilctly to b« found i n fè««tîçfc r««l «stiMit«R ef tb« proportionof iU«« contMinattf om W *«»»ur^ only «t r«U-tt»«ly l«r r«4-i«ti»n <lo««a» S ir *M«pl«, in tbt «ztroia* of JJff «t «1. A 8 / M
t wm* not«4 to«t tk« hi«bt»t 409tof th» eoat«anat«ii proportion was 0.5
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- »t«rms»Uon contamination, • * . I s 'iL. -*
tALLBNTTM »t »1» / 2 V tloe that th» r»»*OB. f *r th»r»»tri«|i»n tt th» proporUé» «
à Mi»*i4»raUo« »f th» «*to*4«BTT» r»Utin« »mar«i pi oportioa / d«nt«MiAat«< fracUoo/ « ido«» / » / »T«r Bnli«it«d T«^O.» »f ». thi» «imf » i i l fmU
to • £•»• irtMr* tto» p»portl<» «ppwaiwi^»f «*t«*Nttta* *f * f ^ f pwiUTW la
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tautr»M»UM U tt»
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thii Mtttfc. »rw tlmtfh ta« p*^U*l tf «*tt« ««nt«iiii«Uo* waftW ttjfr «at I» ynawgtiwi «ltb 1* al»o th« 1—t float yt«ldl»«
tta»a»«a««l f**«t
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irradlattaa kf 2? WAl» 2a aapa*l««xit» taa Mrvitia* «f
doaa yialdio* jtllJ^çaitiYa ftp^it» im Màrah» If «atha aontb,-; of lng»»t In ubiaa 4ha hijbaat «ajft ••!•« «f
th . W ••ti*»-t«dwan mwlxt »f «uryiTtfcg a£«r*»*ga«la«t i f «qatii to JL
f > '
J5ra •" ^
th*
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I f ofof tk»
* radlattoo teM i f to» mbcr of «1«(OM*SPÉXIM» «urrlvln*i»f« «f ctll»
K»
«fta r*p«li>
aber* firm HmU*t*U*m V UmLXtmA by twoFlMtly, th.
fhts •itwtlMi eta
of Ml«rMr<ttl«k» i» not
«rt»4JLotttwr «Vtaatiom mi? êçf»r wtMA f»r I M U M * «MM
- M -
taava*t*atl«al* »r f«<wj.toff oonUlnio* vatfrr «r»Aecordin* t* «XXUUTO «o* HUXTACHABJ18 /50/# inaettafea ««Us«or rtl*«t» «WML f «etorc, iiUUst x»p«ir «tayaM, Into tfcf «wiraa-atat htlp to tit* dma«*4 to l«ch pro«M«
of
HukX i# .*• bi r«pr*Mnt*6 bar*. " " ' " 7 _ • - , . " • > * • " ; J • " • - . . . . - . . . j i ; - _ , • . ,
dois a»t todst, lu tb« p?*ett««
tht
tgrpfftp m—% MMUKHM mâ/ov tfa«ao«t rmditMrt strala
coAtaalatttai th« it«» »rior to th*
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»*,îiÈi'S?ï
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S
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tl«t «omt* of« M
to r*«oh
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ê*?v**n** of th. prop«rU»« «fa
ofI n to
thi»of >2
m - l . M mA %or«r th» »«riod» of
in
jaero©ri«nl»a» n » distributed on product» prUr to th«»c«ordinx to th. l«f-»or«*l «i»tribuUo«, « d
«c»r4t»i to
ijT«li»Uon. «t fttft* ^•4 in •urriviog of «nt tar»* ^«r pr«dH«i «ft«r th«
MLeroeo«ca«
•t t .aU.t t « i l l twpàid to tti* »tr«i».
ifl to th« op«r«ti*M of
«r*
1 I
W *€•*%}*«•* by tut•tan V«1M* «f
* • * W Wfcg /2*5 MrM/ IT th»
106 Ma «M, «aimai 1000 •i«roor«*ii*i» p«r «M pro*tt«t, W I J M U -
git tait of up*, Y«iu*» OP «r««,
prier t»
« U «U
ît »
th« 0MXmm 0.44,
to W
3Û 000 or aox«
of «U «tof mec**, to
b*t«k «r f©r
»f
th« of
fter
th«M «fcttftttt
of
th* batch of on* million*
C* 8 • f • r • n c • e
1. U t r.J., ÏAUamïUl JU? iUtdtytioii » WriliMttioo - iht choie» • /ftwm. Jourtul, • t 216-218, 1SG5- — ^
. , B«tli*OYA" I . l * « - » t » r l l i « U o û <»at*«iii»ttott of
••tion d»««. IOA-SM-192 •K«dl»at«rili«»Uoo of «edloal product*
2*
3. of 1*74,
9.
of BD-
ÛHIlfA O.L.,
ii*. /' *ro««dio«* of
rjofe
C.O.I l»f«l
of-soul» tm«awir
Publication» Ltd, îloûtteal, Canada» 1978
$*»
!>
10.
15.
16.
for the I«ai«UoB StorilUation ofIMA
nni.tilj». C»U Hostow, 1973
oti at
mion E»dl»Uaa St«riU»fcUoo «f M«dlc«l
ÏAÏÀ Ti«ui^ pp.Blologlc«l
3.1
ni j *
of 4iapo«abl«of ro(H»U«u •t.yiUifttlon
product., IJJU^M-1«» "R*iio*1974% IAÏA ltste« pp. 50-5»,
I t .
/ «at.
- ion
A.,«f
rX34» 531-534, X9U
B., UAWtOW) t M OàU. 4 - . t . l
i ^>
1 * *
•"> i
I»
t
* ï
ï *
7 <r > >. _>-
1J. TAttOTTW A» IEAX A.A.t « » * «f tfc» validity of m i l l* J *ol«tU* froq*w«gr of «oataclB»t*4 it«m* and I t r o - i a g r»4-
20» WIT1 / , «f/O.ïu
21. A.t DUX A.i.td««r«* of #UrlU^r tM»r«&et, in,
In
, A.J.
22.or
I.» ft*l«re«yk L.i A« Att«qrt «TBf d«pf of lir«4l*tio» «fc b««« «T th» «ptetr» ofr««iit«>ot. AAte Ml*«»kl»loglo« Polooic», 197»
IA1A
IAIA
24. BBOBtt <3., W.I.»05»» Hp«lo»-
p. 3T-42»
25. KlSSfm* jE.i I*ol*tlo» of rm*&m raUUit'•' '-' * '^jp^fll ~^HM^^>4NW3JPMHMNVNI * Wm~
^ ' • & > * . . -
47i U0Q-UO5, 1972O
^0^; KWU f.t . i
-60
2T» M., IAXAM S . , BSIQA1A S. t»t i i«ol<
•ioroooctn. r«-4* loaa
*t «on atiH«ation «earn indltatror «icrobipXogtqu» 4» 1»da* a4*MC«c 4 * Ut Société <*•
IfT5-
» Control trf th« «iorofaiologic«lcy of r«dt«tl«n •urHi»»Uwà j>U»t» by •««as of B.
15»*t
OjÀ, 8tr. fw«i«i Ajl wâ poli$m
29. C ,•port» B*O113JM
1 liJU•f
Xo. 15»,t
pl a f*ai«tioa »t«riU»«U«A
pp, 197430. of • t r . l a «ad tfct «haie*Th« • • l « e U o »
pr»pa**tion of b io lo«ic»l KMBiton forof
J l . tUMUBJUl M.A., KKOUUCTA fc., OOJUWWA • . . Itoatrol :
i s poliwnareh o.^rialo». / i »ion* as*», i m . ^; ; ,
3a. Julian M., cKi K., IUMAX »,» iunxi»«uoA of
USA
lit,
c «* / •
J4. CH. « *l«robiolo«le«l ecmto*!It» ffftot «n
«ri* «fttr1414
159, in r»di«U<m
35.
36
>
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46. CBIISIBjeWI S.A. r MUKHRJI S*, HO•ontrol of r«di*tt*Q *toriU««tioa of a«di*«lI . HiSil »»p^rt »o. 122, ?«ifc» W
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p a p e r s published cm work done uadtr the Contract
f>
HOBJtovi V., SUUJKX D.t î t s t ing th« efficiency of radiationsteri l isation / i n Csecb/» Acte hyg,,epid.,aicrobiol.n.u 132-136, 1977
V., KUvli R . I The quantitative and quaUtativ« bacteria,ç of tht hunan cadaver «kin i n r*ap«ct to radiation
stérilisation» IAEA Advisory Group Matting, Athens, 6rc«c«, 1976
V., twM P. , SlAUKiC D.Î IxperUnces tdtnradiation «ttril izatlon i n C««choslovakia.» in "Sterilisationof a«dical products tqr ionising radiation", tds* £.E*L* aaughran,A*J* Ooudie, Multiscitno* Publications Ltd. t Montrai, Canada,pp. 119-124, 1978
V., BURlXlJKOVX £ . , K1£N E.Jcf dersjo-epideraal grafts* R«s. in B^periaental Kadiclnt, i npress - 1976
CtttLKOVi V.t OLERICHOVX D.: JÛkrobioloficlcà kontasinae* Kkarsk^cbv^robkû a sterll isaee pomocl ionlzujicitio câfeni / Microbiologicaloonta-alnation of medical supplies sivi thsir steri l isation tymeans of ionising radiation - in C*och / , Sasopis likafù oes-
/Prsha/, l a press - 1976
KÀDIOSTERILIIAÏIO:I CP DSICO-SPIISR^AL GRAFTS
V. Chylkova-Ho:?âkovâ, E. Buriankovd, R. Klen
SVuT - Centre for Research and Application or Ionizing lîaâia
Brno - Veverskd 3ity£l-ca
Faculty Hospital, Tissue Bank, Hradec îlrâlové
Czechoslovakia
3 u n a a r y
ïreaze-dried shin u ed as a temporary biological dressir.-
belongs to grafts in those radiation sterilization seers to be
the method of" choice. Studies have been ainad at evaluation cf
pre-stsrilisatio:i contairiins-tion of si:in grafts and eiTi^iarisy
of substerilization dooac»
It was found out that the pre-sterilization. ccntaixination of
disiiu/ected and fï'eozs-ârieâ al:i:i grafts is favourably lev. Anp
average niunber of 3,43 colonies v;as settled for 1 en * Jilresdy^i1^ dose 15 W/ks did not reveal aurviving or^sni^ns in pract:'.-
0-1 experiments.
Attention was payed to psychrophilic nicroflora. Isolated
obligatory and facultative pcychroyhilic ïriicroor aniG-T-s did net
represent hasard as to the radiation resistance.
The evaluation of efficiency of the niniuun steri l is ing dooe
25 kJ/kg v/ao raade taking into account the biological origin of
the ckin £ruft3 those yield a suit-.sblo nutritional n-ediun for
r.uitiplicatj on of bacteria.
It V/DO stated that the do.-jo 25 ko/k^ {3-ivcs a Ui,;;h ju:v.\v.-.tco
Of oteriJi. ty< Tt hir; been rerwYroM'led to •iocrcaje L'ne n;i:-;i:-.;-.i
jzr;r: i.l.î /,! n.j flo;ss t.f> 20 h!/]:i', vvovi<\<n\ Vac M .:v.\ v1. n.- Oi' -. i ore-
J
- 2 -
or anis:r.s on 1 cm of the disinfected and freczo-dried s".:in
jrafts of human cadavers is 100 v/ith the upper li:.:it 130. The
use of a lower dose would enable diminishing in the deteriora-
tion of physical qualities of derr.oepidernal grafts.
I n t r o d u c t i o n
Ionizing radiation has been successfully applied in prac-
tice serving today as a v/ide-spread arthod for sterilisation be-
fore all of various plastic and rubber instruments and apparatus,
disposable ::.2dicc.l -.rocucts and several ~sdic:.ri2s or ichcr.v.aoouti-
cal base materials. To provide the best chance for patients to
survive it is necessary to. prepare in the best possible -cr_-.=r
the biological tissue for ^raftin^, and such a treatment also
includes a method of sterilisation. Sterility has to be on^urad
with the' little or no deterioration in the physical a~c nochoni-
cal propsrties of tissue. Radiation sterilization seems to beco-
me in ' s ose tissue grafts tho r.ethod of choice (1). Freeze-dricd
skin used as a temporary biological dressing b3lon~s to such
grafts on the first place (2,3). Nevertheless, it io desirable
do decrease the usualy taken aininun dose 25 !:J/k£ in order to
save the most qualities of grafts those influence favourably the
healing.
"he efficiency of the given sterilization xethods depends
on the number and resistance of rdcroorganises contaminating ob-
jects prior to the sterilizing process. Our studies have been
aimed at the evaluation of the pro-sterilisation contamination
of skin grafts and a possible diminishing of the ninir.al steri-
lizing dose.
ï.ï a t o r i a 1 a n d n e t h o d s
All studies were performed on samples of s'dn taken rros
ventral side3 of thighs from human cadavers. Tho skin treated
- 3 -
\.'
according to the conditions Tor talcing of ûeiv-iO-epiderintl ^rufts
(5) was shaved and cut into pieces of approxo 1 cm sise in
the laminar flow bunch.
The prs-sterilization contamination v;as assessed on l'rssh
and freeze-dried samples, those were rinsed in O,lvo peptone
water solution and filtrated through nsrr.brane filtres (Synpor,
50 sa size, average poor sise C,45 /un). Filters were incuba-
ted on plates with Count i-late Agar (Gxoid CM 3^5) for 3 days
at a room temperature and for 2 d at 35°C. C-ros3 colony ;.or-
phology and conventional inicroscooy of Çram-stained prepara-
tions were performed. ;
In order to assess psychrophilic microor-anisic, the
freeze-dried derr.o-epiderrr.al ^raft samples v;ere cultivated for
14 d at room temperature. Isolated strains were further tested
for their ability to grow at 4°C. Decrease in nicrobial conta-
mination was studied on freeze-dried souples of dermo-epicer-
.T.al grafts, those were exposed in a Co-50 source (AECL lyps
J-6000) to doses 1, 5, 10, 15 and 25 kJ/icg. After irradiation
the tasted samples were overlayed by Nutrient Broth (Cxoid Z.L 1)
and cultivated for 14 d at 35 °C.
ïhe niicroor^anis.is those appeared to survive irradiation
were studied in connection with radiation resistance. The resis-
tance was evaluated by determining inactivation curves of bacte-
rial populations dried from phosphate buffer suspensions.
R e s u l t s a n d d i s c u s s i o n
She distribution in the number of idLcroorganisms on sanples
of fresh and freese-dried der:no-cpideraal grafts can be seen
from the Fig. 1. It is evident, that in a ^reat number of sa^rlos
no microorganisms were isolated: in fresh crafts it was 31,7 f.'»
in freeze-dried ones 36,6 (,S. This fact indicates the distribu-
tion of microorganisms will not correspond to the roisconian
distribution, but rather to the exponentional one. The stated
mean value of microbes per 1 cm of skin was in frosh samples
- 4 -
12,83» v«hile in freese-dried decreased to 3»43» The significant
diminishing observed v;as not caused very likely only by i'rosse-
drying, but also due to tlie time of treatment by the uaed dis-
infection solution Septor.ex. V.'hiie the fresh grafts v/ers rir.sad
in peptone water in -iodiately- after taking, the samples deter-inud
to freeze-dryinj v/ere frcesed at a temperature of -20 C.
Caszo (6) in sinilarly organised experiments found the pre-
starilization contamination count considerably hi^har» The dif-
ference might be accc&ted to the way of treatment before the
testing, namely by the disinfection ::.suns used. The favourably
low number of microorganisms in our case is by no rùear.s influenced
by a good hy^ieny in hospitals fron those the donours v;ere taiien,
and probably al30 by the fact that the experiments \vere carried
out in v>i.nter season.
As to the qualitative point of view, it can be stated xhat
in the samples of the fresh -rafts cocci \;ere isolated ,-cstly
(69,GCS), less often then members of the genus Bacillus, Jr po-
sitive .and Cr negative rods and fun^i. In the freeze-dried
grafts a decrease in the contamination by cocci vras observed
and the r.ain part -.vas represented h^r the sporeforning bacte-
ria C64,4;j).
'Us v/ere further interested hou such a lov; pre-sterilisaticn
contamination can be decreased by increasing doses of ionising
radiation. The freese-Gried ijanples of derr.o-epiderxal grafts
v/ere irradiated in a Co-60 source by doses 1, 5, 10, 15 and
25 i:J/kg. As expected single cells would survive the irradia-
tion, the samples v;ere subsequently overlayed by liutrient
Broth to provide a good chance for repair processes and survi-
val of microor^anisr/s. This method v/as r.ore sensitive to de-
tect survivals than counting of colonies employing r.ez.brane
filtration. • :'
The results confirmed, the higher doso v;as used the sr-vallsr
number of tested oaravlea yielded /irovrth of surviving .-.ùcrobes
(see Table 1). If the fraction of tests with positive growth was
plotted against the dose, an exponontionally decreasing curve
- 5 -
was obtained. The result is demonstrated in the Fig. 2. i'rom
510 examined samples 65,8 :i -were contaminated prior to the irra-
diation. After exposure to 1 kJ/kg a fraction of 29,2 SS of samples
containing survived bacteria was detected, after 5 kJ/kg- 7,5 ;i,
and finally after 10 ku/kg the fraction made only 0,7 vï. It has
been observed that no microorganisms could be isolated when
irradiated by 15 kJ/kg or more.
In the Fi^. 3 four inactivation curves of the most rosis:ant
bacteria among the isolated strains are presented. Strains I.ro.,
2S 3,, 21 B 2 and 15 3^ were isolated from tests irradiated by
5 kJ/kg and belong to the genus 3acillus. i'he strain IIo. 15 G
v;as identified as II. luteus -and isolated after irradiation by
10 kJ/ks on examined skin grafts. The counted constants ~z and
n, those characterize the slope of inactivation curve or the
extended.beginning shoulder respectively, and thus the resistance,
do not indicate the isolated strains v;ould be a ^reat danger as
to their resistance, xhe situation can be illustrated by 2 fol-
lowing consideration. Provided the samples ere contaminated by
100 cells per 1 cm possessing a similar resistance aa the r.cst
resistant strain No. 15 3-, isolated in our experiments, then
in order to lower the original population to 1 surviving microbe
it would be necessary to iriadiate by the cose 5,76 kJ/kg. If \:s
like to achieve a sterility degree 10 °, that is accepted general-
ly as sufficient for radiation sterilization, the dose 15,1 îcJ/1-cg
would be needed according to the theoretical counts»
In our studios a close attention v.ras payed to psychrophilic
ciicroflora, importance of v/hich v/as stressed by Kl en and He^er
(7). The presence of psychrophilic bacteria on tissue grafts might
bring complications if such microbes survive the conservation by
the method of "cold shock" and of course by radiation, too. One
strain of obligatory psychrophilic fungi and 9 strains of facul-
tative psychrophilic spore-formoro, cocci and yeasts were detected
on unirradiated skin grafts. Among isolations from irradiated
samples 2 strains of cocci and 1 strain of Bacillus 'showed a weak
v
growth about the temperature 0 C after 14 d of incubation.
Inaetivation curves or those strains in dried stats ^re evids.vt
from the Fig» 4» Their resistance was not remarkable and did
not reach the values of the most resistant ioolated microorga-
nisms those were mentioned aboveo
C o n c l u s i o n s
Following conclusions can be made from the obtained results:
1. It was found out that the pre-sterilization contamination
of disinfected and fraeze-dried skin grafts is favourably low.2
An average number of 3,43 colonies was settled for 1 cm .
Already the dose 15 kJ/kg did not reveal surviving micro-
organisms in practical experiments.
2. It is necessary to take into account that fie biological ori-
gin of the skin grafts may be a reason for providing a suitab-
le nutritional medium for multiplication of bacteria, those
can make clusters. Further proteins, fat and other conpor.cr.ts
of skin may enhance an increased resistance of microbes on
the freeze-dried grafts. Another point is that the- distri-
bution of microorganisms on skin ^rafts might reveal single
pieces with higher contamination.
3« Obligatory and facultative psychrophilic microorganisms do
not seem to represent iicsard as to the radiation resistance.
4. Viruses are expected to be killed during treatment 'JI? disinfac-
tion means and subsequent irradiation.
5. Considering above mentioned remarks it is possible to state
that the dose 25 kJ/kg gives a high quarantee of sterility.
It is recommended to decrease the minimum sterilising dose
to 20 kJ/kg provided the mean value of microorganisms on2
1 cm of the disinfected and freeze-oried skin grafts of
human cadavers is 100 with the upper limit 130.
J
- 7 -
\,
This work v/as supported in part by a research award of the
IAEA Research Contract i-.o. 1589 /R- / R3.
R e f e r e n c e s
(1) KLSJÏ il . , PACAL J . : i.os. :isp. Msd. 1G7, lp-20, 1975
(2) ::L2:T 2 . , PACAL J . , 7A7 .A I . : 7A2A l l .c^rch Co-^'i.'-.'^-Llo:!
:.:eetins on' rcadiaticn' Steri l isst iori of :..edical Products
and 3iolob-ical Tissues, Ij.'-aùoc LrdlovcS, îo.3S, I975
(3) rERRY V.?. : Crycbiolojy 3, 109-130, 1566'
I.IURFI-IÏ P.O., JJALLÏÏ::ÏIRE'A. : IAEA .^search Go-ordinatad ;.33-
tinj^ on Radiation Dtari l isat ion of Medical Products ar.d
Brolo-icrl 'Jizsuas, .Iradec I^i'lovû, J3.3I;, 1?75
(5) i;L3IÏ R.: Securing a;vd 1-r2cerv::.tion of Tissues, SZU I-raha
1957'
(6) GAS20 L. : IA2A Rssoarch Co-C--dinat-3d l-.'eetir.^ o.i Radrliion
Ster i l i sa t ion of i:ed:cal l-rociicts ."md "-jiolojicsl i 'iciuas,
Hradec I'^'.'.lovi, Î33R, 1975
C7) KIS." R., HZCER J»: Acta cl:ir. plast. 11, 4;-55, 1S£9
- 8 -
1. Distribution in the nuober of microorganisms on nativeand freeze-dried darmo-epideraal grafts»
2. Decrease in number of contaminated derœo-epiderasal graftsafter irradiation»
3. Inactivetion curves of four strains isolated fromderoo-epidormel grafts irradiated by 3 and 10 kJ/kg,
4* Inastivation curves of tbreo strains of isoalated
obligatory psychrophilic bacteria.
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