zierg019 hitps:ranslate,googleusercontent.comfranslate_f Ne 172-281 Page 1 NF 172-281 NOVEMBER 2014 ven portal ae tly forbidden (Sunda on ne ceamcn. Alston exploitsion. rpodanion a re-iemination, ver pal attr th form hadcpy oF ther mad) ey poke. Standards onting For: PIERRE LABAT Customer: 80005075 onder: N20150127-1665575-T con: 27101/2015 a 1:20 ‘632ier2019 NFT72-281 ao Standards online the 27/01/2018 at 11:20 NE 172-281; 2016-11 Pagersrintit:LABat NF T 72-281 November 8, 2014 (Clasicaton index: F 72-281 Ics: 11.080.20 Surface disinfection processes by air - Determination of activity bactericidal, fungicidal, yeast, mycobactericidal, sporicidal and virucidal tuberculocide including bacteriophages E: Methods of wb dno f ses - Detention of std, gil Yeti, year, bled prc ad viride ining baterohagss erbateriien,fngzdenlevrszden,myhobkaiide,ohetalcden pores oven Abe Bakaophapee French standard homologated ‘yds ofthe Chie xcstve fer of APNOR Correspondence sss eof psietion of ts cre her tnt mak irl or Eugen cori esig wa he me a summary TW doen descr amet for dtr the dfs of sues diction process bys sed in te bal sear an, erty. gh, nail and oman hogh pysielprcses snail. “Tis mato make i poses search, nde codons loe to he sic, the sty roe infect we fer the ecton of fte by a, whether or oot reed (ine oa uo ea-arateplenenton wiht pretece amar cectd ast to mal process in te presence of heap, descriptors Intention aha Tene: DisctANr, DISEFECTION DECONTAMINATION, ENVIRONMENT, EFFECTIVENESS, ACTERICIDES, FUNGICIDES, SPORICIDES, TESTING, ANTIBACTERIAL ACTIVITY, ENOMBREMENT, MICROORGANISN, ENVIRONMENT modifications Cepeda cnet, compe reson sd wt of dee tne oi ae canges bre ben made of aty ida myctnnel dtin iererg bs nd madame tod oeentog he oclio e pr (851.2, a8 sume he of tetany arf epi (ped adeno pe ntpsciransiate.googleusercontert.comitransiate_t 21632ier2019 NFT72-281 ‘ferme cules ahh peceenst Cha Pht, corrections ARNOR- Allah reseed Standards online the 27/01/2015 at 11-20 pageigPintit: LABAT NET Standard ‘The standard is ended seve a. asf slaionsbotwoos econ paras, sii, techni na soca ‘The sundatd by satis vlusry Referenced in a enact, its binding onthe pases. ‘Regulations may make al or pat of stundad mandatory ‘The standard ls a document developed by consensus within a standardization body by soliciion of representatives of al imerestd partes. Ts adoption is preceded by an naury Pabhi, ‘The standard i eviewed epulaly oases rlevane over ine Any santa is deme effective fam the dae om the is page ‘To understand standards “The ears steaton i drawn tthe following points: ‘ony verbal forms must and must bo usté express one o ore routes ha must be respocted io comply wih this document. Thess reguremerls ca be fond in the by of he stand rin annex deur "normative" For test methods, the use of the itive coespunds to to arequirement ‘bxpresion ch s, ti appropriate and itis recommended ae wed to expres possi refered ut not equied to comply wit his document. Verbal forms ean and ea ae wed to express a useful or nommandatry suggestion a tip, or authorization. In adition, this document may’ provide adtnal infomation to aia tae ndessandng ore of erin elements o to cls their apps, without ting to respec, These elements are presented in he frm of mates oF informative annexes Standards Commission {A sandadization commie rings tops n pveo fel of stil he necessary expertise to the developmen of French standards and ench positions on dealt European sanded or Ineraatina. can als prepare experimental standards and books Ifyou wish co comment on tis text make proposals for ehange or petite ints revision, late comact“aominfo@feorny ntpsciransiate.googleusercontert.comitransiate_t erson of 201 NF 172.281: 2016.11 31632ier2019 NFT72-281 ‘The composition ofthe standardization comente that prepared this document is ven Helo. When an expert presen an organization dierent ro hs pare organization thi information appears in the form: paren oxgrizaton(epeseted organization) Fb, Standards online the 27/01/2018 a 11-20 PIERRE LABAT Antiseptics and chemical disinfectants NP 172.281: 201611 a NPT T2281 AFNOR T72Q ‘Composition of the standardization commission President: Mr STROHL Secrest: Mr THOMASSIN - AFNOR es squat ntpsciransiate.googleusercontert.comitransiate_t NSIT CH CROHOLODIALE SARL loge sas 41632ier2019 NFT72-281 ston Iny-NS TUTE REC AOCRAMIOL OGL SAM Fi, Standards online the 27/01/2018 at 11:20 jooiietir ABar NETTR281 - ‘Summary enor se eennil 1 Held of apleat 2 Normative references a ee et 4 Prescriptions ‘est method, Precip Preliminary validation esto th absence of sida etfs. Test lf Marine and reagsate Micr-organis ofxt Culture moa and regents Sepportsof ger used Apparatus nd glasware General ‘Usa ecupment for microbiology laborry and in particular Preparation an crumersin of test suspensions Preparation of test suspensions (@acteria, yeas, molds, pores and mycobacteria). Prepartion of ssponsons in intern bans ‘Enuneratios of suspensions prepared in the interfering substi Preparation and enumeration of vr! suspensions Preperton snd enumeration of et pensions (@actral host and bacteiopbage suspensions) ss Procedure for exlusing the ety of he process NB T72.281; 2016-11 Page 10 10 a 2 au 25 » 30 s Procedure for evaluating the activity ofthe process according the conditions of use tansmted ty the manurtrr (aster, yess, mois, pees, vite, ‘uceriophages and nycobactera}. ‘Vireidal et k (oe F3) B and bacteriophages aod mycobacteria) iE General [Enyrerstion of colonies sd iis 9 be respected EE bucterophage and phages (54) ntpsciransiate.googleusercontert.comitransiate_t ‘Experimental data sd calelatons (aster, yeasts, mois, bacterial spores Detrmiaation of te rumber af gen inthe sizbil suspensions, 7 Procedu for evaluating the mnyeobuctrisial activity ofthe proces: description in Annex F 0 51632evz01: NFT72-281 eerminatin of te numberof T gems on he contol aes (5.5.1.2). Dutcmiaton ofthe values stad nthe prclanay et 913), _Detrmiaation of te umber of germs and plagues on the et media ae sortact the log reduction aia (58.183) Rests interpetation Test ep Be BEES Fb, Standards online the 27/01/2015 at 11:20 Pagé’7PiFRRE LABAT ‘Summary Annendix (normative) Preparation of Bacllas subi spore stock suspensions al Matra an reagents hore defined in 5.2 and he lowing ‘Aa. Bouillon (TGR) Az Agr(Mvay Anns (ommtive) Distances dsinfecton device- germ carriers tobe used Annex C(acrmatve) Test Schemes Annex Doormats) Summary table of est conditiens und objective in function activity aims (formative) Specie methodology ee ‘nomircted ultraviolet adit sinfection processes nL Gener a Evaluaion method na Ress terpreation Appendix E (normative) Procedure for the evaluation of mycobacterial activity Materials ad reagents Micro-ogariams of test. Cltace media and reagents Aparatus inerfering substances. ‘Mycobacterial teat uipension Proce frevalusting the mycobacteria activity ofthe proces. Sorvival indicts of provi on suppor, Preliminary validation est for th absence of sida fist. Test ef Ctelations aed intrpeation of esl Clelations see Aisi of his dostment Results interpretation ntpsciransiate.googleusercontert.comitransiate_t to the evaluation of antimicrobial fia Preparation and enumerstion ofthe test mycobacterial sspension in the presence Enumeration and incubation ofthe test suspension othe presence of interfering substances. 58 4 aL NP 172-281: 2014-11 NPTI28L Page “6 6 46 46 ” 8 2 st 6 55 56 56 se 6 “6 56 8 58 9 o “0 0 a 61632ier2019 NFT72-281 Tb, Standards online the 27/01/2015 a 1-20 NP 172-281: 2014-11 PIERRE LABAT NET R281 Foreword This document desenbes «general mthod for 1 ve under standardized conditions lose lo practice, tha the rack ropoted forthe lsinfection of sues by ae achieve te purpose fer which they were designe 19 camare in eproducible codons the various processes betwen the “0 provide an expernienal seme within spose iit when pret conditions move way font thot ven the ext lowe ‘This document aks into account he dnt of on-porus surfaces But ot that of ‘his document applies to processes using aesiveigredeas or physi gens a are wor canbe aomatic, semi-automatic ox manual ‘This paper proposes wo approaches opefarence evasion éepening onthe mode of application processes. We dstingsish -nnrdirete proceses whose objective is the dsnietion ofthe fee of he ate space lo be ete “They ae ofen assimilated to an automat or sem-automaticimplortentation without human presence Jn this eats he document propotes a nigue simple position representative ofthe civerity cvittaton and location of surfs unde fel codons and allowing simple comparison nd objestive processes. The microbial inocuium s positioned ata defined distance fom tbe \sinfection and oriented isthe opposite dizecton. Guidance, spd additoal locations ae possible depending on the intended we ofthe proces. Urvilet radiation proceses srsthe saeco mposfeanex (Annex js Dat nek of experience wit hee processes, Annex Eis informative, dees the atetin of ters and evaluators othe influence of dtnce, exposure the UV soure, ag inthe case ofindect exposure, he properties ofthe fing walls tothe sample Directed processes implement by aplication on surfaces facing the dfasion stn {often asinitae to manual processes in the presence of the operator. In tis case, the inocu is plod ‘acing the broadcasting syste belongs othe manufacture: 1 specify the init fuse and precautions of ue ofthe proces; ‘To ensure thatthe est conditions of the document ae representative ofthe recommended ute (0) [eeovers the sectors f aplication 1) human heath, 2) veteran, 8) agofood,indstil and communities. The test conditons and efficiency objectives are adapted to each of he above ares. ‘The tess deserted in this docomet are based on he measurement ner the specified contons, ofthe log reduction ofbatva, eal, mols, beter spre, bateophages ot ‘ruses of diferent species and mycobuctvia. The range of micrergarsms chosen may prove tobe ‘oo small for some applications, stmaybesipplemenedhy other sans bated on the cxpeineotal scheme deseibed in hs documcet, by varying the conditions accotding tothe needs ofthe ntpscliransiate.googleusercontert.comitransiate_f 71832ier2019 NFT72-281 (62) petal aplication () enisoged ‘The purpose ofthis documest so reproduce he practical conditions of atbome disinfection; ‘mandatory condition are defined accondig lo the ters defined below: For some paramles suc as the dimersios ofthe test oom, he humidity level and the temperature, so-alled mandatory contons "Complementary wil ave o be care ot ifthe ntended se x prefreblyoutside the enon andar basis. Addon cottons te als propose, ‘The tes seperti speeily and summarize the conditions wader wil het at etd out ‘The document quae the poses tha: ist say all he components and consumables eguited tots implementation, Asan example fo hemisal process, the materia pou areas inseparable ‘The process are gerry plemented ser the cleaning procedure and are then tse, according othe sca of appli, in conditions of eealinss or low lve dt. Fr some applications the manure rsommiendatons for vation methods in the presence of ther Intern substances ae ao posible under alional condition, Fb, Standards online the 27/04/2015 at 11:20 NP 172-281: 2016-11 Paga’gPiFRRE LABAT NPT TEs Introduction ‘The purpose of this document ie odssrbe a method for determining the disinfectant activity of maths of dsncting sures by air unde specific experimental conitons. ‘The procete involved include thot ivalving chemical disinfectants in dapesed ot steam and tos involving physical agents, suchas UV. (Arex E informative). ‘This document doesnot tak into acount poceses whose mode of action is based on Soaking md or onthe conic seit or with a mechanical ation by wiping The elect sbove res 8 lsplaceret of alo part of th micoorganisns, outside the surace ofthe support as defined ithe document without being inetvated. As result for procsses that under el conitons of ecumbine a mechanical action wih the dsiafecting action, only the disinfecting action wl be vase This document inches mandatory conditions tone mundstry condition ad ditional conditions (ee $51.1) adapted othe various domains. Mancatry conditions complementary measres humid the onal conto alow te ute of relevant nci-oganianso tain, in erative t those describod in he mandatory conditions, in the cass of lange volume evs (s6e NOT of the paragraph $5.11 ()) Eauivalene with the compulsogy stains they replace, however, mst be previously esablishod. low for valuations oti the mandsory ranges of volumes tmpertare and ‘The skimmed mi add othe microbial suspensions play bth the le of prossie and intererng ssn, ‘The document allows for file germs to dry, and forte ater oly (among germs curt mandatory, only aeruginosa it concerncd) possbibty of increasing the concentation of protective material lint os on dying (fe 8 6,5224.1,5224L.1 (@),$224.12 1b), 52.24.15 (a), $4.21 and Annex D), 1 Application domain ‘he method describes insnded to determine the disintoing activity of the processes sein he sects) haan eat, 2 veterinary 3) grsood, nds and community through physical processes sede ‘The procesesconcemed have the objective of dsineeting the sures ofthe enti space itcding external suraces ofthe equpmen contained in these premise. Airteatmeat apd product or processes specially intended fer the dsinestion of metal eves ae excluded fom the cope af pplication af this document 2 Normative References ntpsciransiate.googleusercontert.comitransiate_t 81632ier2019 NFT72-281 ‘The flowing documents, in whol on pat ae normatvelytferenced a this document etd xe eseaial is application For dated references, onl the eon ted applies. For the undated velerenss, th net eon ofthe Repstraon Document apes clang ay mendes). [NF EN 12359, Antvepieand chemical difectonts- Storage of test microorganisms sted {forthe determination of bactericidal activ (Legionella inluded),mycobacercdal, ported. fangietda and ruil (nluding bacteriophage) (laseaton index T 72-185) |NPEN 13610, Chemical ssictants- Quanta suspension est for ewalaton of ical ati sesint bacteriophages chemical dinfectants sedi the field of fond and agriculture (the industry Test Method and Requirements (Phase 2 Step 1) (CsiticationThdex: T7288) [NFEN 1476, Amispcs and chemical dsfecons-Ouantiaive suspension ts forthe evaluation of irda actin the medical eld Test method and prescriptions (pha 2, tsp 1) ( sJascation: 72-188) “pagan 0015 07020 NP 172-281: 2016-11 FERRE LABAI NET - [NPEN 14675, dnusepis and choca infetans - Quantitative suspension test forthe evaluation of the virucidal acti of antieptice and chemical diinfctant ard in the eerinary fl es ethods and prescriptions (phase 2 sep I (Clesseaton index: 7 2-184) [NEE 1688S, dnteptie and chemical dvifctans - Application of Eropeae Standards om antes and chemical disinfectant (clasifition index: 72-900) 3. Terms and definitions For the purposes ofthis dacs, the ter and definton given in standard NF EN 14885 a well a the fling ems and dfiitions apy a pysteal process rmathod sing a physical agent for example UY (Annex E, informative The metho icles the source transient the aye of geneaon and dissemination. Sea-hased process a ieniing radiations ‘uo ot taken into secout i this document 3a hema! proces proces in which the active igre i chemical agent ffs in gaseous, gud or solid Frm, The process ‘clades the chemi and the associated isin system tht cas not be evaluated separately ‘The process comprises inseparably the chemical an the dion system proces for the dxnction ofall sufuccs incu in a given volume, whatever heir ‘nenttions. The prpae f the processes concered i 1 infect the srfices of the nie space This ype of process is gencally implemented without human presse n the Wace wea a directed disinfection processes rmatods fr disinfecting surces direct expose tothe difsin system or the source. These processes ere maneuvered by un operator who is present in th oom during the cyte ‘The procese ate dtnguthe without preaure (sal spry-ype sprayers for example) wich posse (lrgr appaazs With ane and compres o "seas ntpsciransiate.googleusercontert.comitransiate_t 91632ier2019 NFT72-281 a5 Alsinfectant activity of x Asinfeton process by alr csv ofa proces capable of reducing, by destruction the rumber of couablemi-o-organiss in he onions ofthe document sd from specific sins longing tat eat one ofthe sever OMS oF rmirooraniosbaceri, acter spores, Yeasts, molds, han Vrs, baeropages, and myeobastia under defined conditions and afer deposon on a nonporous erence median 1a the case whee the process is bea testo oe met the nein ofthe dovumeat oly ot part ft peice recomended, he following actives shoul be specified: bated sci, porch acivi, ‘ungicidal activity, yeas acy, veda activity, vse acvity against bateiphages, tuberloidl att and mycobacterial civ. 36 brite germ at drying cording lo the speci eitera af this document, when telson dying greater tha 1S log at te end the drying phase, outside the exhibition peiod anor Standards online the 27/0/2018 at 11:20 NP 172.281: 2016-11 Paget fitkkt EABAT NeTT228 4 prescriptions ‘The desired redustion ale ar expressed i devimal logit ad depend oa the Self application, They are ined inthe tables in Appendix D ad snared below: Dace! ati: edetionsrenter than oF eg oS om smparave text maa ‘tw nonexposc conte! samples forthe ve hate strain used (oce5211-5212-5213-521.4) ~sporcdl activity: reduc greater than or equal o 3 othe st eda compared 79 ortvol media not expose the proces, forthe spore acti stan uted (see $2.18) fungal evi: reduction pester tha o gual oon the test media compared to ontrl mea not exposed othe process, fr the two fang sans (east and old) implemented Gee 5216-5217) yeaa ctv: rection greater han o oyu to 4 on he test media compared a ontrl mea not exposed the proses forte yeast sin used see 5.2.1.6) viral activity: rodston goat than o ogul to 4 on comparative test media vo onmtind-eerened contol, forthe vrs sin 0) plemented (see 5218-5219- 52.110) vical city aginst bacteriophages reduction geste tha or equal 94 on test media compared to contol media not expose tothe process, forthe sain (6) oF bacteriophage (9) implesnented (eee $:2.1.11- 2.1.12) -niyebsctrcidal activity: reduction gear than equal to on the et media compared with otrol mes not exposed the roses, fre mycobacteria) plemented (see 52.115 2nd 52..18). ‘Thete stv canbe determine independ These seven stvities mast be determined under experimental reference conditions defined in 55 appropriate «bated, poi, fungicidal, yeast, vrei! Guan vine) ativigy specie haceiophage and mycokactercdal agent ean be detrrned under other eonons of uation of conte, ature ofthe suppor, miroorgnisns, volume of the es ber gro, emperaire, in ocondance with 5.5 0 take to acount the conitons which erespond tothe spec wc to which processes ae intended ‘These sddonal conditions must be described, emmarzed ad reported nthe Ls repr, ntpsciransiate.googleusercontert.comitransiate_t 101632ier2019 NFT72-281 S Test method Principle SAL Preliminary validation test for the absence of residual effet ‘The peoliminary test cried vt inorder o reveal ny resid] avy to eds af snfectnt vaneported by cris, to ubealreme(n agaand on membrane) and nda method to eliminate it Taus, during the actual testa hatercidal, fangsidal activi ‘sis sporcdl, vical lading aps blenophaes and rue mycobacterial i determined and ota sinpl residual nkibitory elec. Exposure of usconamieated, gas o dispersed moti Recovery in 10 a. (20 mfr viride) of ste Hiquid and sourch for bacteriostatic effet (oc figisatic or inhibition of spore germination or rection of ell sens) due 1 lsiofctnt ined on the suppor, which coud ave ait ation in aa tedium and on the meres ‘ering ordre the dnl por Standards online the 27/01/2018 at 11:20 NE 172.281: 2016-11 Pagenairtkt tABar NET T2281 =10. S12 essayittt From suspensions of microogarisns, 50 pL. deposit onan net carer prepared as indicted ins23 Spread a dy the inoculum ak described nS 5.1.21, thon sini he assembly under defied conditions In conte: wih the product dusedby the ested method Recover bacteria, mycobuctria, yeasts, mols, pores or vrses oF bacteriophage by serpin, ting and uleatonialy treatment, posbly, rom he supers, in he gu reoovery - eomering baci, aycobactrs, yeast, molds ad pores, ition aad inlsion naga of ‘sto the recovery fig. fnebaton and enumeration of developed colonies. Filan on momar ofthe reining recovery Uiuid an rise to linia the disinfectant as much a possi “Tanse of the membrane oan agar medium, Inelsion ofthe medium in agar medium. Incubation and enumeraion ofthe colonies oained, ~cnerting viruses, dilation ofa atin othe reuvery gud and placing ells in the presence of ells 52 restving Incubation and determination of he ia tile Material and reagents 521 Teatmicroorganiams Depending a the atv sought andthe tee of pplication (sce Amex D) al orp ofthe stains rt be se, ‘They cn be obtained fom collection centers. Is 2) For haces = 5.2.1 Pewdomanar aerugiosa ATCC 1S442 ~ CIP 103-867; £52.12 Stphplacacus auras ATCC 6538 ~C1P-4 8%; ~$2.15 Bntercoceus irae ATCC 10841 = CIP 855; + 5.2.14 Escherichia coll ATCC 10836 = CIP 54127 (0) For spore activi: -5.2.15 Basis subatsspores ATOC 6633 = CP 52 @, ntpsciransiate.googleusercontert.comitransiate_t 111832ier2019 NFT72-281 (6 For fingieial avy 52.1.6 Candida abicans ADCC 10231 = 4872; -52.1.17 Aspergis brasiliensis ATCC 16404 = 14318. (For yeasty: = 5.2.16 Candida albicans ATCC 10281 = IP 4872 «) For vised nett = 5.2.1.8 RnleovinusBouie Tipe I (Enric Cysopathogeni Bovine Orphan Vis = BCBO) ATCC VR248, ECBO growa.ce B cell ne cell ATCC CRL-139) or atc appropriately sensitive lines: -52.19 Murine Norovirs strain $99, Pedich Loser Tstit, Bern, MNV gross on RAW 264, cells aree tiB1; #52. Adenosine pS, adenoid sain, ATCC VBS, Adeoviue grown om fel cll aes lineages with appropriate ses For vn aviy agus bteriopbages 5.2.1.1 Bacteriophage of Lactovoccs sti subspecies lst POOL (DSM 4262); 5.2.12 Bectriophage of Lacincocous Its subsp, Lasts POS (DSM 10567) “pope ese ae Ne 172-281; 2016-11 ERRE LABAT one NPT 2A81 ‘Te maliplicaton af hase wo phages mus he obtained fom dhe following hos soa: Lactococcus lace subspecies lc E72 (DSM 4366) (@) For mycobacterial and/or berclacdl activi 5.2.1.13 Mycobacterium avium ATCC 15769, 5.21.14 Mycobacterium tse ATCC 15755 adit est nco-rguaams aroused, hey should be incubated under conditions of optinal growth temperature, me, smospbere,ezvrnmeat tat wil be mentioned ia he fst report, "he adional tet micro-organisms selected do nt corespond tothe specie species, thee bility to provide the necessary inocula must be verified these est microorganisms ton ac not isd bya collin enter, eset exabish their characterise encanto, they must be referenced anand by the esting Inbortry or the est center, ational oleston fr ive yeas 522 Cultere media and reagents 52.21 Culture media for bacteria, yeasts, molds, bacterial spores and mycobacteria 52211 Oreriow All he asses of chemical substances nated in his document corespond othe any rous sis ytd forms cam be used br the required masses need to be adjusted to reflect ieee in eolecuar weight Reagents ust be of analy grade and /o eet microbiological aces. They must be ‘eft toxic or inibitery substances fr test micro-organisms S212 Water ‘The water must be fey istiled on gle und a0 deminer, Sterize hy autoclaving (3.2. (8). NOTE | Ste isi water require forthe preparstin of sspetsions, On the tan, i a sways to elie the water we, for example fr the pepo of ute media WG te es nested NOTH? Inthe sence ofdsillod water of appre guy, wae fr injstione may be ed ntpscliransiate.googleusercontert.comitransiate_t 121632ier2019 NFT72-281 S223 Bacteria count ager tsptone soy agar: TA) ‘To cary out th counts of clvabl astern ($21.1 10521 “yplone, pancreatic digestion of asin 150g Soy peptone, papaya dgsstion of sy Aout soe sacl 508 Asie 150¢ Watergs 1 000. Strize hy autoclaving. Aer eriizaton the pH ofthe medium should be equivalent 6 72.402 fr ane measrement 20°C, ‘Adda nemalizer ofthe disinfectant ifnecessary in the medium (ction iia the PV), S2214 —Srin maintenance agar 52.1.1 195.2..4s0@ 52.2.1. without newralier “pageants mama 20 NF T72.281: 2016.11 i} NET T2281 a 52215 Dilbert of icra rupensions 52.21.51 Bacteria, yeas, molds and pares “Tryptone-soiv chloride ston composed of Irypion,panceaedigention of casein log Sodium chloride (NaCI) ase Water (3.222) 57 1,000 mt. Strilize in th autoclave [5.3218), Aer sterilization, the pH af he den should be exuielent 0 70 02, fora measured at (20 #1) °C. ‘The diet shall ot ease inference Between test mico-organsms and he product sntectnt tested 52.21.52 Mycobactra Solon of wyptone sodium chloride, (80 $22.15.1) Or distilled water (0082212) ‘The die most ot case interference heen et micr-oraniss an the rod Alsi tested. 522.16 Maltexractagar (GEM) For counts of ule calls of yeas and molds (5.2.16 and $21.7), Malt cxtact (cecal quality) 3008 eae soe Water ge 1 000 ‘The malt extract must be f fod grade or example, Cristom powder, marked by Dif) orf valent quality, no: highly purified an ot contsing only maltose rat eau musket by OXOID).») Steric hy autoclaving. Ae sterlzation, the pH of the medium should equ 29°C, ent to $6 02 for one meatureeet [Adda newalizer ofthe disinfectant if mecessry in the medium (meation iin he test reper) ntpsciransiate.googleusercontert.comitransiate_t 131632ier2019 NFT72-281 S227 Medium forthe maintenance of rains $2.1 Gand 52.7900 $221.6 without rewralizer 52218 Fld for germ recovery and membrane rinsing Compson: ‘Tryptone pancreni igetie of css log Sodom shorde ase Polysorbate $0 508 Watergs 1,000 mt Preparation: Dissolve sodiua chloride and Tryon in wat. necessary, add eutalzer speci to isinfestnt under sty Prepac the dient in arge volumes and ive nt suitable ootiners, Seize by atolaving. {1 Ths information provided forthe convenience of ses oft doment and nt ited col conatite an ABNOR commiment otis produc. Equivalent produce con be swede show thas hey ead 0 the same ress Tb Standaas online the 27/01/2015 a 1-20 iBIERRE LABAT NE 172.281: 201611 a NPT 281 52219 Reconstted skimmed milk Ut kines milk powder, gusrantod without antics and withou ative, ecnsted a rate of 100g for 1 sine water Steve for 50 minal (15 43)° Cor S mina (121 3)° Cor erie hy membrane iron 22 pL. 52.21.10 Agar for enumeration of culvable Mycobactera (trains $2,113 and 52.14) See Annex F (51.2.1, 52.211 Medium fr spores fran $21.8) CGincose aga with ye ents. Foremimeraton of ible Bala subiie pores Amin ais, without vitamin, obisined by acd hyolpsis of ain one WO Soluble sarc log. Glucose, 258 Yeas extinct soe FeS0« ole Maso4,H120 0.0001 g Ae. 50g Watergs 1000. Steriize by autoclaving. Aer sesiization th pH ofthe medium shouldbe equivalent to 68-+0.2 for one measurement 320°C, (Other media maybe wed for pores: soe Append A 5222 Culture media amd reagents forthe preparation of viruses (tains S2.1.8 10 82.110) ntpsciransiate.googleusercontert.comitransiate_t 141632ier2019 Sterlizeby autoclaving Aer stenizaton th pl ofthe medium shuld be NFT72-281 52221 Growthand conservation media (Use Eapo's Minimum EssetialEavronmort (MEM) or ouivalet,suptemeted bya concentration inactivated fetal cal serum free of mycoplasma, and if necessary antibiotics and oer prow fctors (Gee NFEN 14476 and NF EN 14575). 52.2.3 Culture media and reagents forthe preparation of bacteriophages (stains $.2.1.11 0 $21.12) 52231 MI? alr rah For the mantonance ofthe hos: bactralstrain, the rutiplictionofbactrophages ad the preparation of the diluent, ‘or phaps: Pepto phytane (hm sys Nour s00g Pople plypepone (om casein animal esa s00g Powdered bet extract S008 Yous Exact 2508 DO) -lecose s00g Arcot aid 0508 Pesotum phceroposphate 19.008: Magnesium spate, 2 0 02555 Water 460 522.12) completes. 1,000 mt, i Standards online the 27/01/2018 at 11-20 pao aBiFRRE LABAT NET R28 ou Jen 107.002 for me measurement 20°C, ‘When fhe M17 oth isthe dunt used to prepae the neualze (0 NF EN 13610), item secessuy to use ‘MIT broth prepared in double concenrion tat is tbe caentation should be docbled fall the ingredients that are added tothe 1,000. of water 52232 MI? agar background agar) Beiround agar forthe qui sie enumeration ofthe ys nes (range) cach blend fom = ony inesious patil f bacteriophages, fone i the bacterial capt of te hast hateriua. |Add 15 g of ga to 1000 ml of MI bet, Dist the ag by bringing io bil while by sting continues Sterlize by autoclaving. Aer terization th I ofthe medium shouldbe equ 120°C. Once ent to 7.002 for one measarement apa: bs cooled (47 1)°C, add 10m of ee Ls Ca 2 slion (ee £22.36), Mix gemly and pour 15 to 18m of agar ito Petri dishes, $2233 Overlay agar (surface agar, sof agar) For cnumeration of bacteriophages: Disolve 65g of gain 1000 of MA beth (Gringo a bol while staring contsuousl). Spread the supercooled gar is et tubes (2S ma to 3 mL byrabe, Stile hy autoclaving 52234 _Diluentfor phages (bared on % dated Ringer's soltion) Tappa the seis of ations to determine ttt of phage sspensons enameraton of ys zones by ages: Ringer solution died a sodium chloride 22508 ntpsciransiate.googleusercontert.comitransiate_t NP 172-281: 2016.11 151632ier2019 NFT72-281 potassium hl 0.105 = anhyseous clea soride 0.06 8 sodium bygogencatonate 0050 water Gee 5.22.12} make up 0 1000 mt. Ads 10 of MIT elie ra 9 mf died Ringe soon Sterlie hy autoclaving Belore ute al | mofo C2C12 sk oaton to 101ml. rth Aion NOTE Ringer saan ca be prepare oa eat-tose bls cng 1 the comendone ath spi 82235 sMotamp For resuspension and intact preservation of page pails Tis HA 2ae mc S85: Mgs04, 7120, 258 voter (6 $22.12) complete ot 1.000 ma, A se pl ofthe ber fo 7401, Seize by acting, at standard enn the 2701/2015 a 11-20 NP T72.281: 2016-18 Pagan pintkr Labar ss NETRA 52236 CaCI ochotis (1 mol/and 0.05 mol/L) Dissolve 11099 g 015.88 ganhydrous CxC2 a water (Se .2.2..2 and make wp 6 1000 for bain th tsk soliton a 1.0 ol Ta 50 moma, rexpetvels Strize by eitovlaving 52237 asic acid olution (witha volume fraction of 10%) For the acidification of low-fat ilk forthe pueparalon of eid whey. Dilute laste cid ther solution with 90% volume ation ia water (see 52.2123) to bain a working solon ofa voimefntion oF 10% To do thi ada Spats of water 9 pa ofthe selon Strlize by asocaving. 52.24 Interfering substances 52241 Orerion ‘The interfering subsance (9) must be Selected according tothe application aes ofthe proves (s= AppeniixD) eit al postbleto ad shined el for ceain fail gems (og Preudomonas ‘seruginosa is adition the recommended iterrng substances. The inerfring substance (6) must be tee) an prepared (10 mes concentrated lative othe final consenrason nthe est Prepacation and sterilization methods and composition shouldbe noted in the repo 2.24.11 Conditions fr dsinfotnt use in human medicine 2) Comtiton of cesniness orbactricial nycobactercda. mgd, yeastcdal and spoil activites, the ceases eontion i represented bythe use of skimmed mika deserted in 52.219 an uted to 1/20 whi its the ss to Aryng for mrorganians orgie gems defined i 3.5 among the obligate germs only Peradomonss aorupinaca i ntpsciransiate.googleusercontert.comitransiate_t 181632ier2019 NFT72-281 concened), afer plementation of fist est unr the condtons of the document for bacteria and result wll he poset inthe test report, wo methodologial adaptations are proposed far taking imo account te fos on dyin its pemisibe to increase the quant of protective meri up to 1/5 wof recone skimmed milk inode 0 Timi the lous on dying (quay of on-viahte germs) below 1S log The iil ee ‘minimum and the log redson target required to claim confomity remain the same to thotof he etegry gars, espestively 6 log and Slog for Pseudomonas aerino is permissible wo increase the intl inoculum o $10» 4 at olan og te on the supports witness afer drying During the tests both msinods wil be cried out in parallel. As soon a5 one of he wo ress is postive, the activity ean be elimed oth rel, howe, should be incl inthe repon. NOTH Inthe cate of rite perma define ie andi he cae ofa incre he inca, = my ef den ger i ld tobe prteiveonkeiig ter rovidd by be dont (Copenh geet ed sppbewton we) thus een tered ta ae et of del gos can vert he gn lea as onto oi rere wie at lo be oberved ace ofan sete it he nese in sid ik For vrei sets appropri tows commercially availble bovine bum (Con fsetionV) snd prepare ita follows Dissolve 0.3 gof bovine albumin 100 lof water complete with died water pt the mak; Steilze by membrane lation of 0224 keepin the egertor fora maximum of ene month; th ial concerrton af bovine albumin (BSA) inthe tet 03g Toe Standards online the 27/01/2015 at 1-20 NP 172-281: 201411 (ERRE LABAT NET T2-281 “16 Dir conditions (ovine serum albus) Serum albumin bovine Ini appropriate to use bovine album (Coa faction V, wept 1 the microbiological nods) vale ia lead and prepare as Flows: Dissolve 3 go bovine albumin 100m of water complete wit died water upto the mat Stlize by membcane iain of 22 the Sal conssnteation of run Bove sbumia (BSA) athe test 3 g/L stop nthe refigertr fora pao fone moth Sheep erytirocyes eile defibrinated sheep blo avaiable commerily should ewe, -centfige erythrocytes fom a leat 8 of rsh defibrinated shep bloods 80 @ fo 10 eit afer removal ofthe resuspended supernatant rom enthroeyte in the ler (5.521) Repeat his prose at es tre times sta he sepa coloess Solin of tovion aan eel sheep erthoytes: Suspend 3 ml of concentrated eyhreytesin 97 ml of 3% (w /¥) bovine albumin sobson; se 3 convention of bovine albumin ad sheen erythrocytes the test procedre is respectively 3 / Lat m/s “inorder to avid contamination, tis mixture should be divided nto the number of algun regis for oe day a ep thers in separate contines fora manu of 7 days 22° C10 °C. NOTE Theprapntio ted onda in he rte fina miemay roa pee prt anda ntpsciransiate.googleusercontert.comitransiate_t 171832ier2019 NFT72-281 ‘imum ering stance diferent lm tao the formation of agrees of blo cll In at se HBevane ss shou bSaken me scnaa me alone “ 52.24.12 Conditions for dvinfectans ed nthe veterinary Geld 19 Low-level sil condins (bovine albunsin ston) Dissolve 3 gf aston V of bovine slbursin in 100 of water 100 volume sk. Complete vith disiled water up tothe mar. Steilze by 22 membrane firation an soe at rafigeratr fora mx of ne nth ‘The final concotraton of bovine abun ding th tot it’ g/L Low-level sol conions in he presence of sized mi: For agile germs as defined 7346 (ong he obligate genns ony Preudomonaraeraginoa i oneered) so conbitons saat) Dissolve 6 faction Vof bovine albumin in 90 mL of water ina 100 mi volumetc ask. Complete vith disled water upto te mat. Steriize hy 022 membrane filston and sores refigerator Zor masimum of one ment, Mix volume for Volume with ecastued skinned nik (522.19) ‘The final onsentraton of bovine albumin ding the et 3 g/L. a the reconstuted skied miki ited 1208 «© High evel sil contons(siature of ovine album solution and yeast ext) Dissolve 50 gof yas exes in 150 of water ina 250 mL volumes ask and Teve the foam {ll Fil wih water up tothe mark. Transfer to a clean bole snd steve sutocave Allow colt oom tenperae. Take 25m ofthis sluion ina volunetic ase 0 ml then aa 10m. of eter, Dissolve 5g asin V ofbovineaumin in the soliton while sing and alow the fosmn to edo Make up othe atk wilh water and steriie by 22 w membrane Seton, Keep at rofigortor (2° Ct 8 Cand use during the month. eee ee rans ih20 NE T72-281: 2016-18 Paget Al 0 NPT 72281 ‘The final concearaton inthe test 10 Lf bovine album and 10/10 yeast exact, [NOTE Prepantion ofthe higheve it condone presence of im mi my rete protocol ‘este and aioe at eet inerering subse 10 or reasons of soiity af icgediess snd eiph concen ofthe ok clo, Inthe cae, scot ald he ake ofthe ine ats 52.24.13 Condions for shaft used inthe apriod, nda and domestic tars, nd ination 2) Comtions of lesniness Forbactericia, mycobactrcdel, ancl actriophaicdl, east and april ative, he cleanliness conditions rpeseaed by the use of skimmed milk a desclbed in $22.9 and dated 120 a which iis the drying os fo mieo-organisms. For agile germs defined 3.5 (amon th obliga germs only Pradomonar aeruginosa concerned) ee conditions ia $2.24 1.1 (a) For vnuial activity is appropri to use commercially avaliable bovine albumin (Coniston V) ad prepare its follows: dissolve 0g of bovine albumin 100 of water (82 $22.12) seize by membrane flkson; keepin the eigcator fora maszau: of ne ath the inal concentration of bovine albumin (BSA) inthe teste 03 g/L Dissolve 0 fstion V of bovine albumin in 100 of wale 100. volumesrie Fillup tothe mark with water Sterlize by membrane ition of 0.2 ‘The final concentration of bovine albumin in the teste 0.3 g/T ad concems he vrs ntpsciransiate.googleusercontert.comitransiate_t 181632ier2019 NFT72-281 i conditions Dissolve 3 of astin V ofbovine amin 109ml of water 10 ml voles flask ill wil ited wate. the math, Strlize by merbranefvaion of 0.2 wand ston he efgerator for a maximum of ne month ‘The final concentration of bovine lbuminSring the tests 2/L ©) Din canditon inthe presence fskinimed ilk: Fr faite ge ak defined in 3.6 tong he ‘obligate germs only Pseudomonas aeraginos is soncerned) Se conditions in $2.2.4.3.1 Dissolve 6 fstion V of hone ahimin 90 mi of water ina 100m volumetric a ill wi ited water. the mask Strize by manbrane ftation of 22 wand stor in the efigerator for & maximum of one mont Mix volume for volume with econ skimned mil (5.2.21). ‘The final concentration oftovine album during he ets 3 g/L nd the reconstituted skimmed milk sted 1200, 52.24.14 Interfering substances specif o bacteriophages 2) Wey solution For the test conditions, prepare an aid whe solution ftom skimmed mi as flows pasteurized (1.59% ft content add 0.3 ri. of lc acid slution with volume ation of 1% (ee $223.7) 4010 moi shi, mix end hod the samp at 00m nips fo 30 min. Mix of tne time ring this 30inut prod. Then sediment te mik pots precipitated by cctitagatin at mama speed (400 g minions) for 30min. trie the superna hey) by rmomirene iltacon (porosity 045 ym and store a4" Cto8C, to obs a working solution of woe action oqo 10%, requted a substance intron agent requted for phage esting, dle a orion te acid whey solution with nine pan of wate. Store the whey solution ta volame faction of 10% at 4° Cto8* Sra encase NP T72-261: 2016-11 agerabetit LAB NET R281 <8 ‘Whey soins should be stored between 4° Cand °C fora maximum of one month Fr periods of shel it, hey shold be kept roan tempersties bebe 18° C snd 20°C, or lower For the et prostate, the ial concentiation ofthe whey sluon sl be qual toa faction olume of 1.0% (6) Stammed sie or the test conto, pepe the recoasttutd skinned uk (15% a const) of the following way -reconstiite sim mi powder, guranteed fe from antibiotics ar ative, tthe ts 9°10 1 of water (ee $22.19), ~stelze wih scam tee successive days (30 mln cach ay) a 100 °C and eave tom enperatre ‘otwoen tam resiments not eve inthe refigeator betwen seessive reatments wi steam ‘Unite skin ik sdf matsn te host bacterial sin eis alo posible to size w (115: 3)°C for 15min. To asin working solution of volume faction equal 10%, required a subtance optional intering agent fo phage testing usa portion of singed mile wih nine parts stele wae (see 5:2.2.1.2). Store the skim il wth a 10% volume ation between 4° C and 8°. For the ext proce, the inl earn of the skim mi must eel to. volume faction oF 1.0%, ntpsciransiate.googleusercontert.comitransiate_t 191632ier2019 NFT72-281 523 Supports of the germs used ‘niy se new maa foreach et S231 Reference materials ise in tiles ts! L4901 (NF EN 1008-1) fn 3 cmt 4 em in dimer, grade 2B acording 1 the requtements of [NP-BN 1OOR8-2, bier! Ssh 2 Surices should be sft as possible win A sine stel gauge of 1.2m or 15mm and are sed only once and hen ined Before se place the sures for 60min in a beaker (minimum capacy: 50m contsiing minis 20 ml of Decontt 8% (Rinse he supports under a steam of fesly dsl wae fr 10s Daring the subsets preparation plus, dom llo the uric o dey it any way. The supports mst ‘nly be handle using pier, Rinse he brackets with water fer another 1 secon to ease cpl emo ofthe surfactant oder to enre x saisfactry water flow, ts poasible owe a pressure vese eiverng the stile Mud hough asuable hose and fitings or any other method ‘nd atthe flow to provide approximately 2000 ml/min. To dite pace the clesn de ‘na bath containing 10% (0) sopeopanol for 18min. Remove the suppor and dey ity evaporation der Taminar sie fow 52.32 Additional supports Any support maybe used provided itis non-porous and described in stalin the est sport ‘Toe supports mst allow to spread tertna 30 pi inoculum ona surface ofthe ode of cm 2 (2 em of ameter. Che the fessiilityo spreading on hs suppor. They mest th be able to be nrodued ino container as 150m o S00 ma. and ten ia te Poti dts for inclusion. Clesnng and sterilization sul be determined acordng to he nature ofthe support and specified in 2 Saintes tet ics can usualy be obtained Ina from manufacturing ampanic Pid Standards online the 27/01/2015 at 11:20 NP 172-281: 2006-11 HERE LABAT ar NP TTDI 53° Apparatus and glassware SAL Overview Striize ll plassware and equipment cmponens that come int contact wih culture madi andthe reagents or sample, excep hose provided stile, by one of he following methods by mist eat by setoctving (5.3.2.0): by dy hea, in aot air oven 532.1 S32 Usual equipment for microbiology laboratory and in particolar S321 Steric 12143)" for ()Fordy best stsilization «hot i ove that canbe sinned at 180°" C +5 C for ‘ime 30 in, at (170-£5)°€ fora erat (1605) °C frat least 28 5322 Waterbaths Thermostat bath cooled a 65 £1) °C. 5323 incubators Bacteriological neahaor, velit or unveiled, a eatin temperate of 87.1)°C for ntpsciransiate.googleusercontert.comitransiate_t 201632ier2019 NFT72-281 shying in less han 2 hous of eontasnaed media “Incase or teri atv, which ean be maitsned a (361) ° Cor (37-41) °C. nouhator (fo yeast spovcda, iuedal and ued epainstHacteiophages),wbich maybe kept at(30#1) °C, = CO sincubatr (54 ais, 5% CO 2), capable of being maintained at (36+ 1) Cor (37-41) °C fa nebaton ‘oll ulus a vrs. S324 pH meter with an aeursey of+0.1 pH units at(20#1)°C NOME Tomearethe plo te apr mel, enewton eta ofan cletode should be wsed fact 5325 Stopwatch 53.26 Rlectromechanieal or mechanical sirer 532.7 Membrane fitration devices, consruced ofa material compatible wih the subtances ‘The appliance ust have fine with @ working volume of at east 50 mL. mast be suitable foruse with ler 47s to SO rm in date nd OAS kero in prod ‘The vacuum source wed must emu regular leaton ate In order a oan an even stun of ‘microorganisms onthe membrane and avo 0 long atin, the device shouldbe st for ‘er 10 fring gui in 20 104, Refrigerator canbe thermostated between 2* Cand 8 °C 5329 Graduated pipetes witha nominal capacity of 10 m2 mL, | mL and mL. Pipees utr elibratrs an he we, ‘3.2.10 Petr dishes om 90mm to 100mm in darter and 55mm for membranes ‘3.2.11 Glas bends (danse: 3 mo 4mm) 52.2.2 Sintered gat ker: porosity bstween 40 mand 100 gan (180 4793) = poroiy 2 pra Standards online the 27/0/2018 at 11-20 NP 172-281: 2014-11 Pagerank: tABar 532.15 Cenfuge capable of an atcleration of 2,000 g (0008 minima: NeTT281 -20) the cas of bacciopaes) 5.32.14 Roux fas with a stralght neck 3.2.15 Analyial balance of adapted scope 582.16 Spectrophotometercqupped with a monochzomaior saaur cum pep (rani Mow at 38 2 3/ bial vacaen<75 eben) S218 Sterile filter membranes with hydrophobic eges with «poo of 45 ym ameter 47 mn to $0) cellule eters or any other materia suitable fo the product under et. 3.219 Device for keeping the media avay frm the source 3.2.20 Microscope preferably in phase conta (for spore est); invesd microscope fo cain Vnuidal assay, 53221 Clamps ntpsciransiate.googleusercontert.comitransiate_t 211632ier2019 NFT72-281 5.8222 18 mm or20 mm test bes 5.3.2.2 Contalners: ves or vil of adequate capacity 3.2.24 Conia con 1, Wide opening, 280 mF SOD. 5.3.2.6 Via wih screw caps 5.4.2.6 Pipettes wit single-use tis, delveng 0.05 ml. ~ 50 for noc deposition 53227 35 Kir altraonie vibrator 5.32.28 Magueti ster to keep cells suspended before iocustion 53.2.9 Pete digs, sterile est bes, cule Masks of spraprit espucty 5.42.30 Volumetric Mas calibrated at 20°C 5.2.31 96enell sterile microtiter plates, sical right wel plates for el clare anvils eal eure ‘5.42.32 Am ee cream machine or commensal ice cream for cooling the cll nd reset nists during the tet, 5.1.2.8 oe bucket wit ce nd water 5.32.3 Biosafety cabinet, as Il (anleo be uted daring the dying ep ‘noculums) 5.3.2.5 Freezer witha temperature not exceeding 10°C ‘5.32.36 Desicator rth vacsum conto manometer othe we of PSM vented 36 eraive ‘oe the dying operation of incala on suppor 3.237 Hlgh sped homogenizer: se Annex F (13.1, aio Standards online the 27/01/2018 at 11-20 Pagarzaitkt LABat oa S4 Preparation and enumeration of test suspensions Preservation of est microorganisms and parent cultures ‘Test miroorganiams end ther pce caltres must be prepared and preserved in acordance wih requirements of NP EN 12353, Working eure of est microorganisms S421 Bacterial and nga srpenions 2) Bete: {a orderto prepare the working cutie of he test ria, trnepla om the TSA agar stock sae and incubate at (3741) °C. Alter ISh to 24, preaze a second appl inthe same way rom Sit subeulu and incubate or Lo 26. A thin ansplaing canbe done in there way om the scan [NOTE The secon and/or ibs the woking ep) In his ese, prepare anothe24-hrsuulire beloelesting, Do nt perform a oi ranean, ntpsciransiate.googleusercontert.comitransiate_t in mo posible to prepare a second wanplant ona patil dy, iti arise lo we subelare “48 hous for subsoqunt subsulting, povided tht subuting was matin inthe ioubstor fr 48 hous NE 972-281; 2016-11 NPT T2281 221632ier2019 NFT72-281 Inthe case of ational stains, any deviation om his method of colturing bacteria of Prepraton of suspensions shouldbe ted, initing the reason inthe test rept Incabate a7 = 1°. 1b) Modan yess In oder to prepare the working culture of Conde alicoe, ransplant rom the sock elute 1 GEM aga nd Inoubatat 30-1) °C. After 2 to 48h repre e second wesplnt inthe same way fom, St suber and inate or 42 ho 8 thin ranplaning canbe done in the sre way to ftom the seca NOTE? The second aaa ed abut woking 6p) "itis not possible to prepare a sesond wansplan ona particular day, itis pemisibleo use a subculre "7 wur or subsequensubcltng, provided ha subcltring wat maintained in he custo or 72 Bours In his case, propare another 48-brsubeute efoe testing, Do not perfor a four wanepa. For Aspe brass use waneplanting on GEM in boxes of Rous and inesate a (301) °C evento rine dy, $4122 Spore suspensions Spores arent anspanted but are red a «tock suspension ce Appendix A. Mocobacterial usponsione For the preparation ofthe working culture of Mjeahacterum avium and Mjcobacertum trae sce Appendix F e212) S42 Preparation of suspensions in interfering substances S421 Sesins $21 1.2.14 acter) ‘ake 10 mb of len: (52.215) and place in 100 mi vial wih S geass beads, “Take the working sure wih he handle and ans the spl to he dient 522.1.) The eel shoul be suspend inthe dient by immersing the loop and ubbing against he val ofthe wa to detach the sls Shake the as for 3 min with» mecanial ser, Aspire the suspension hats wit te glass bends and transfert to another tbe TOE Standards online the 27/01/2015 at 11:20 NF I72.281: 2016.11 iERRE LABAT NET T2081 2 Adj she el number ofthe saponin with luce (5.2218) estimating the mabe of wis bby means ofa spoetopbotomter or by an appropriate means Addo this suspension a propriate at defined in 5.2.241.1,$2.24.1.2 and 52.24.13, the kine milk to obtain a elution ofthe ter Tyo the interfering substance to obtain ition of his lastin 1/10 The ial ceri ive ould be Between 5.107 and 2.109 CFU/ml, For file germs as defined in 3.5 mong th obligate germs only Pseudomonas aerupinasa is concerned) two methodlopical adaptations ae proposed to ake nto account the loss on dyin: iva penile wo increase te qutatity of protective mil upto 1/5 of recontitated shined il inode 0 lini the foes on dvi (nti of noa-vible gor) below 1 Tog. Te ini le ‘minimum ad he log reson target required to claim conformity remain the sme to those ofthe gems ofthe category, fr example respectively 6 log and Slog respectively for Pseudomonas aeruginosa itn pease to increase the ntl inoculum o 5.10 940 at obtain «log i on he supprts es ater drying Daring te tes both metas wil be cri out in pall. As soon as one of the wo ess is postive, the activity canbe elimed. Both reslls, however, shoul be ineladed in the repent nis upto he ahorony carrying out th ett ensue the assurcy ofthe titles obi ing tests prelinay ntpsciransiate.googleusercontert.comitransiate_t 234632ier2019 NFT72-281 Store thi sapension in the bath a (201)* Cand ue it within 2 hours ois preparation. 5422 Siuin 215 bacterial spores) = Spore sock suspension ofthe test san: Bacillus subi pore stock ospensin is prepared inthe testing laboratory in aecordane with Annex A, = Spore ts suspension: to prepare the spor test suspension, late the tek surpension of spores with ue $22.15 Aldo this surpention a sppropite a defined in $22.61. $221.2 and 52.2415, be kina milk to obtain a ution ofthe later 1 za o the interfanng substance te obtain alton of his Jasin 1/10. The inl porte shoul be betweon 2.10 and 5.10 CFU mf. thems to lnbortory crying out the est to ensurthe scarey ofthe tiles obained ding peeliminary tnt Maina the ts suspension inthe wale ba at 20.4 1) °C and se within 2 hous Microscopic examination under magnification of 400 should fe performed immediately ale preparation and st Defoe the al Wo Bight e dsence of vegetative ell ad germ spores. the presence of germ spores> 1% the suspension shouldbe removed, S423 Siain £2.16 (Candide alicans) Take 10m of eluent (5.22.15) nd plein 8100. vil with 10g gas bead ‘Take the working cure wih the handle and rans the sample to he ivent. es necesary to pt the cli ‘suspension nthe diluent by immersing the hall and bing against the wall ofthe Mask to detach ‘ells, Shak the flak for min with a mechanical ier, Asie the sspesion hat ith the las bead and ane anather ibe, Adj the nurra sl the uapension wing luat by estnsting the numberof us by means of a pectopbotomte or by at appropriate means ‘Ado this supenion asthe case may eas defied in $2241 1,824.2 and $2.24, the skitumed ile ‘monde to obtain ation of he ater 1/3 or the interfering substance in onder buat tion of the lterat 1/10 The final ite of Candide albicans mast between 2.107 ad 1.108 CFU ml, Jn is upto the sbortory crying out the ett ensue the ssursey ofthe ies aban ding tests prelinay, ‘Store this suspension in the waterbath at (20-1) °C and set within? hours fs peparation “panera 7eransa20 NE 172.281: 2014-11 NPT T281 S424 Seain 52.1.7 Aspergits brasiliensis) Suspead cells fom th working culture in 10m of sterile plysobat 80% slain 10.05% (m/¥ Jin water 52.2.5 Using serie gis spatula, remove the con frm the rice ofthe culture. Tranter the aspension nto conical ask and bake gel for 1 mini the presence of lass marbles, Fier he suspension trough snered fie. Microscopic examination ver magnification = 00 inmeditly afer preparation for sake sre 1) the presence of high concemtion (atleast 75% of ehinlae spre) af mature spores characters, tht, ehinlte spores (a opposed to smooth spores) [ste Figur land Figur 2} 2) he absence oF spore germination check a: least 10 es); 3) inthe even of spore getminaton, reject the suspensions +4) absence of mycelial fragments (check atleast 10 Fel). myc s present, cay out a second snared Slaton ‘tke mye i il presen, reject the apenion ntpsciransiate.googleusercontert.comitransiate_t 241632ier2019 NFT72-281 Figure 1- Photo N° 1 Observation of Figure 2- Photo N°2 Observation of conldlosperes sing an optical microscope: centration of mature spores features with echiulate appearance (adequate suspension) NOTE ‘Theimpsiliyof taining 15% oF exhale spre my be due the culvatn of Aspecis bsesis ‘mat sed to pode tee spores. nti as itis necessary ows tp from aot ello of tales and/or ing mallextact ap: for ate epi ‘Then ajut by adding storie water (52.2.5 the mumber of spore ia the suspension by estimating se ramber of colony-forming units by appropriate means This es suspension mast not be stored for more han 2 days ata empeatre beeen 2° Cand 8: Mix the test suspension immediately before use fo resuspend the sores. Adit this supension a the ese my em defined in $2.21 1, $224 1.2 and $22.41, the skimmed mi in onder to obtain aston ofthe ater 1/ xn the interfering substance in nde to obtain «ition of the itera 110. The final tite of Aspergi brasilnss shouldbe betwee 510 a 1.107 CFU mL, [nis upto he abortory caring out the est to ensue the scarey ofthe ies obtained daring prelimi tes S425 Souins $2.19 and 5.2.14 (Mycobacterium avium and Mycobacterium terrae) partion of test suspeason: See F213 of Amex F FRO Standards online the 27/01/2018 at 11:20 ygeRRELABAT NET T2081 04 43 Enumeration of suspensions prepared in the interfering substance S431 bacteria Enumeration of acai test suspensions: ute th adjusted baci supensions Gee $42.1) 1010-6, 10-7 and 10-49 dent 5.2.2.1 ‘The count ate cried out in pall by ichton NI and N2ftaton Inclusion: Collect a sample of 1.0 ofeach ition in duplicate end inoculate by incelation 11 dep. Using pipet, ntodice cach 1.0m serpent separte Ps dishes nua 1S ml to 20a of Supercoled ISA Agar and eooled to (48 = 1)*C forthe bacteria. ivan: Colleea 1.0mm. N2 sample in duplicate frm each ation and afer each sample 1.0 mina searte membrane fitation device (5.3.2. Fier immediately Fike by doing rise (5.2218) inthe same manne a nthe ts (5.5.13). Then drop each membranes on he suc of the gat medium (8221.3) or bacterial ssi, incubate ples at (96 1)* Cov(27 + 1)°C for 24, Spread all bones on wich its aot posible 0 cout te eolonies for any reson. Incubate ntpsciransiate.googleusercontert.comitransiate_t NP 172.281: 2018-11 251632ier2019 NFT72-281 bones 24 hour re. Do not cout colonies on bones that a anger bave good colonies scparted, Recouat them on the ening boxes. {LetNI be the numberof ones obtained by inclusion, and N2 the numberof clones by itation which wil be cared aut impart $432 Yeasts and molds Enumeration of test fungal suspensions; Dit the ated nga suepenions (ne 5.4.2.3 and 54.2.4) 40 10-5, 10-410 nent 52.215 ‘The counts are eed out in parallel by incision NI and N2ftraton Inclasion: Collect a samp of 1.0 ml of ech istion in dlicate and novela by inoculation INI depth. Using a pipet, intodiee cach 1.0. emp into separate Pet dishes anda 15 mL to 20 of supercooled GEM agar an coted (45 = 1) °C forthe fang Filatin: Collect 1.0m, NZ sample in pice from each ution and tant each sample 10 ina apart membrane firation device (53.2: Filer inmedintely Fier by doing "nse (6.22.18) inthe same manne as inthe ts (5.5.43). Then drop eck membrane on he srice of the apse mem (5.2216) Ja the eae of Aspergllu rasions, divide the sample nto wo, thre oF fur sive portions pproxinately equa nd waster cach partion wo a scarce mombane Staton vessel (63.2.7, which amounts, fra dupe sample, incu fou, si eight membranes. For fingal sin, incubate plates at 30 1)" C for 241 Candid albicans) and foe 42% 2 8 (peril Brains), Spr all he boxes on whic it 0 possible to cunt he celoais, for whatever eeason, Count the bone nd dtemiae the numberof closes forming ust Jncabat the bores for another 24 hours. Do rt resount olanies on boxes ttn longs nave cones well separated. Recount them on herring bok For Aspe hraioni,ontinue incuba between X pm ad 12 pr and necessary, another 20 hours 2124 hours, povided thatthe number of colonies (sll clas) increases. {Le:NI be he number of olonies obtained by inclusion, and N2 the aumber of colonies hy iration wach wl be cared out in peal Bor Standards online the 27/01/2018 at 11-20 NP 972.281; 2014-11 TABAT Pager2irkir NET 281 S433 spores For enueation of ta spo testesiag dont ($221.5), suspension, pepe ditions a 104 and 10s of the sutpenion ‘The counts re cried out is parallel by acuson NL and N2 stration Inclsin: Collec sample of 1.0 mi of each ition in dla and transfer each sample 1 mi on separate Pt dishes anda between 12 nL and 15 en of gluco agar with yeast GGL (5.22.1.10) in supercooing cole to (451) °C. Fiatin: Collect a 1.0 mL. N2 sample in duplicate from cach iui and want each sample 10min separate menbrane filtration device ($3.2, Filler immediatly Fiery ding ase (5.2.2.8 abe same mane a ithe te (5.5.4.3). Than dp est rmemirane on the sr ice of th gsr medium (5.2.2 Incubate the Petri dts a (301) °C for 3 days. Determine the preset number a aon for sch bor, lela the surbsr of CFU/ml inthe est suspension. ntpsciransiate.googleusercontert.comitransiate_t 261632ier2019 NFT72-281 1LeN1 he the nuber of colonies obtained by aclason and N2 the numberof clos by Kilvation which wil be caied aut impale S434 mycobaceri For counting th est suspension See F2.2 of Annex F S44 Preparation and enumeration fv S441 Suspend vires stock ‘The vrs mus e mui on lange sale to obiin vrs suspension with these srstrisics thar the ference wl suspension For sear ceaans, only 10 passages fam the cial vias arcallowod, For altpcaon and preparation of vrs suspensions soe 55.2 ‘The vil suspension is stored in aliguotsat-70* C or preferably at -196* Cin guid mitogen S442 Viraltest suspension ‘The stock views suspension i sultiplid within a suitable celine end capable of producing & high concentration of va prices, Cel debris is removed by cenifaton (40 during 1S min) This preparation i called "est vis sespension” ‘The est views suspension i sed without lation athe tes ‘Vira suspension must have a te of between 110 7and |109 PFU, o ust be high enough ro that at leas a ile rection of 1 sane dstermine, {in exceptional eas, he vrs nuspension my be concentrated sng appropri methods Ge uraceitugation, [NOTE | Toavoid he formation of grep, sonication my be nesesy NOTE? Totes te quliy ofthe virus suspension, an eraton of the roti Ge Lowry’ meted my be esormal for beer epee [NOTE 3 thei ofthe vil upension ast eins acous he os cased bythe ying topo te supp ostrinate (5.5.2. and 352) “pagh ain tigae ™ 7mransan20 NP 172.281: 2014-10 S443 saaaa NET 226 Infetsiy est Cyopatie fet ration ing dation method) $4443.11 Tirtion of vrs on cells suspension in mrs pltos ate vial suspension with ution at 1/10 jn a volume of 0.1 mi fv uapension + appropriate volume feature medium example: MEM + 2% SFV). Pipe tips must be hanged er ech ison step, Trane. mL of each distin ox orcight wells ofa microsite pte, starting with bigest dition, Aad .1 mf of ell culture suspension at deasy tat allows for Formation ‘fa monolayer > 90%) in a est 2 days, Six or eight wells serve as a celular contol and receive no ‘inl suspenion ‘The viel eytopathic effec read using a inverted ixoseope afer thea sopra incabaion peso as el (depending onthe type of vi) ntpsciransiate.googleusercontert.comitransiate_t 271632ier2019 NFT72-281 '4.4.3.1.2 Tintin of vrs on pre-established monolayer in microtiter les ‘Transfer feck dion 1 si or eight wells microtiter pte coming celle contact 90%) a monolayer witout ay medium. The lat vow fix a eight wells ecives 0. a. of ‘alte medi (MEM ~ 2% SV) and serves a cella onto |Aftee Lh ofncuation at 37°C, 0.1 of el eure medium iad to each wel andthe pes ae incubated ot 37°C foun appropriate pio. The viel eytopahic effect eed wing a miroscope revered $443.13 Range meinod Wells of plastic plates surfasediamete 30 mim 0 3$ mm) with confi cll monolayer ae washed once with phosphate bulered saline (PBS) and inoculated with 02 eof seca ditions of tae ius in culture medium (MEM 2% SEV. Thre wes re gery used by blason [After an adsorption pesod of hat37°C, daring which the monolayer cll moisture is rani by iting the anes every # min to 10 mio, the inocu is removed ane he cells in monolyer ae wathed once with phosphate bateed saline (PS), Sobsequenly, the wells ace covered ith 3m ofa mixtre oF 2% menage oF aa uitble semi-solid medium and MEM concentrated twice with &%6 SEV, The clues ae meubsted for 2 days at 37° Cin a CO 2 ineubstor (se $3.23), The lame ranges ca be counted ter incorporation of cond layer (2m. avin these composition athe fis and also containing 5% of a solution of seta (1/100) ad incubation dso in tae ck at 37°C f0r 24 to 48 hin 3 CO 2 acaba eee 5.3.23). (Counting en kobe peered after aon nfmethyl violet Monolayer cll ae fed ding 2a. of 10% wihlooacesc aid (TCA) tothe gat ayes for 10 mites to 18 mites room temperate. The agar layers thea removed and? ml of 0.1% methyl woe s added to 20% ethanol. Ader {0 mi at 1S min at oem temperature, the wel re washed thoroughly ith wer and Isisbouces (white spt) ae counted. NOTE CP teint en be wed a an aerate a he age met Ti Standards online the 27/04/2015 at 11:20 Paga’2girkir LABAT n S45 Preparation and enumeration of test. snd bacteriophages) spensions (host bacterial suspensions SASL Stock culture of host Bacteria Inocalate the recone skim milk with «volume facon of 1% of culture in gud media or with loop of bacteria fom sloped or canned ML" agar, iacabate for? hat (30-1) *C and tock clr ofthe hos sain n resorted skimmed ml the eigerator set at a termperatie of beewoon 4° C and 8° C. Witt a span of wo Weeks, let tes ok cops grow throughout overnight at 301) * Cand repeat th procedure to obtain a esk stock etre Wit ie neceseary to proong the preservation rove the cultures in he skimmed ilk temperatures between «18° Cand = 20° C or lowe r,s eerie cultures ntpsciransiate.googleusercontert.comitransiate_t NP 172-281: 2016-11 NPT 2281 281632ier2019 NFT72-281 S452 Working culure of host bacteria In order to prepare th working ule of tb host bacter,trnsplan fous the tock culture it he both of MIT elt Gee $223.1) nd ineabate a(30-= 1)" C, Use a Volume tion of 1% of noel ora act ina guid medium ora loop of bacteria om an MIT agar sloping or boxe. [Aer I6hn0 24, propa second ranspant fom the Sst in the MIT broth (volume aston of 1% ocala) apd ineubate for 16h a 26 hat (301) °C. From ti second ane, you cn perfor tht ansplasthe sme way Te second and/or ‘he cid tunsplning continues he working culture or cultures. it sno possible to prepare she second transplanting ona particu dy, is permissible to prepare he second Transplanting rom 2 te wansplan, provide ta hs ben preserved afte a 2h nebaton 21301) C, in aveigetor se ata terperatue between 2” C and 8 C. a this case, before continuing, thi eaneplan shoul be prepared nd incbated 16:00 0 26:09, Dono proced to a oth trast 5453 Stock suspension of bacteraphages nates be, wld 0.1 mL of the reference pags iste 0.1 mL of a working altars cur: host bacteria (else fom 16026 hin the MIT elle rt). Supplement he sample wih mmol/L CaCl by adding 0.05 mL of sec 50 met L CaCI tan ston (98 $22.36), ‘Vortex briefly aod neat for 10min 3 00m temperate (le between 20° Cand 21 °C) or allow the dsorcon of phages or bacterial ells. Ado she test tbe 10a. of MIT bot (pebeated to (30-+1)°C) mix ely and Incite at (50-1) °C (ee 5.3.2.3) ati cell Ise appears. The growth of host bacteria (Ge turban lyst should Ye followed To he naked ee, or petesably by measuring the oa density of de eltue wih stable specvopotomete settoa defined wavelength eg 20 am. Prepare conto est ie iia, but replace he phages ste wit 0. brah MIT culture (60e 5223.1). Compate the tutidity inthe est abe wit that ine contol tbe Afr si ofthe Bost bucvain the test tbe contining the ages, ps the supernatant contig the pages ona iter membre (045 ym porosity). Store phage sock suspensions at 4° C10 °C tl ws. Pid Standands online the 27/01/2015 a 1:20 RE TABAT. NET T2281 28 sas Suspension of high re Bacteriophage From stack suspensions of bacteriophages (Ge 5.4.5.3), prepare decimal ditions nthe divent {or phages (See 52.234. To do this add 1.0 of stock phage suspension t 9.0 of docet ‘or phages. Mix and prepare te following diusions in he same way until dtton 10. Coie sample of 0. ml ofeach uon in dupa and transfer each .1 mL sample a separate est be, Add 03 mi ofa werk bactrlcltre er 4 pnt dh, which has developed. ‘nthe M7 eure broth, Ada 0.1 mL 50 rao CaCI sltion (se 2.2.3.6), mix Iefy an inubat or 10 nat rom tempat allow adsorption of phages on the cell atrial st, ‘Add 2. mL to 3.0: of M17 Oveiay Age (6€ 52.2:3.2),previesly supercooted ntpsciransiate.googleusercontert.comitransiate_t NE T72-281; 2014-11 201632ier2019 NFT72-281 {a boling water at of in a icromave oven a then ood ina waterbath at (47:1) °C. [ttetiy mt an then for spread th saps to pnes conan backgrond agr MIT (ee522.22). To obtain ahomogencous dsuibuton ofthe samples, tbe boxes mus be tilted slowly by hand etre sliifcaon ofthe overly agar, (Cae mathe taken prevent an sncontlod overeating of the oven rom bing excessive and unconolled liquid agi. Unscew te caps ofthe cantines to pevent ay sk of explosion. Inaba plates a (301) °C frat least, oven for 16 9 24h, azine the pits ove the appearance of yt zones due cea an waar phages (pcs) i the bacterial caret [NOTE 1 PIO8 pgs page ave sa edges anda dancer | ato 2am Phage beaches UO at lng 3 rn dlat) nd sounded by tidal (NF EN 13610, (Choo rm sod plates fom casade ions hose wih confluent yi Con these boxes, the Beaches must touch ech other and a hin urpet of Yerainngunysed ell must be huey ‘se. Do not choose hones oo which the ceil erp as Been completely removed a on which the nvdal beaches do not touch ach oer (tha i the beaches re ally ole, because itis not posibl to obtain igh il sates tom shes boxes. Clie the phages fom te aga plats tat eval conn! ysis by seeping the sf aga with see angled plas rod, and rane theo a tube ora icrosentige vial: int each ish horaughly wih Sm of SM bulls (ce $2235) and pour thee hules nto testes eeniige) cowespening tthe Boxes. ‘Shake the tes gon rom tet ne for a east 15min t oor temperate Agia sot agar and cll debs by cenfgaton (15 mine 4000 ‘Toe spernatant must be Siteredthroogh a iter membrane (see 5.3.2.7). Phage fom the same strain bt harvested fom different hoes shoud be groupe together fore dktermine the ig ofthe phage suspension NOTE A phage sate propiredaconigo it med ily fe of protein contain gees re ofthe page suspen soba ae bone «10 ee 10 PF Store ig tire haceriopage suspension at 4° Cto 8 °C for 3 moat (2) Confluent es generally acre on ogar plates inculted wih dia ranks 10-107 10-« Tok Standards online the 27/01/2015 at 11:20 NP 172-281: 2016-11 HIERRE LABAT = NET 7228 For enumeration of igh ite bacteriophage suspensions, prepare ei luton inthe age dilvent cee 5.22.34), To dois, 4 La. of ie ite baresonbage suspension increased to 90 ra of the phage dunt. Mix and prepare the following dilations in the sre Way nl iron 10 Duplicate 01 ma sample the 10. and 10 tions and pour each 1.1m sample in aseparte out abe. Add 0.3 os 16 hour hater eltire 124 hin clue both MIT gee $44.3.1.6, Add 0.1m of 465 nolL CaCl? solution (ose 522.65, brielly mix and inet for 10min t oom temperate to alow aorption of hago om bacteria ost ll Add2Sm-t03 eof MIT Agar Agu (se 5.22.3.) previously supereoued in ‘boing waterbath rin msrowave oven and then cooled in water ath a (27+ 1) °C. ntpsciransiate.googleusercontert.comitransiate_t 301632ier2019 NFT72-281 ify nix td then uniformly spread he apes in plates consining agarose background M17 (sce $22.32. none: to obtain + homogsneaus dstibution ofthe srpls, itis necessary to slowly boxes by hand before slficton of he agar plate SASS Preparation of text bacerophage suspension Before applying the test procedure o determin he virial activity ofthe products, the phage the high ice bacteriophage sesperson (se 548.4) eased between 8.10 # and 3.109 PRU Ja with SM tafe (se 2.23.5) in ede obtain the Final est bactesophage suspension for implement the procedure Before proceoing with he act test, check the acta fre ofthe phage suspension by preparing ris iuions ofthe test bacteriophage suspension using the phage diet (ee $2.24, For hit Todo this add 1.0 of the tes acerophage suspension to 9 mL of phage diluent Mix and prepare the lowing dlatons inthe sae way unt ion 10.7. Coles a duplicate sample (1 ma of ution 10 and 10-7 and taster cach 0.1m sample oa spar tt be, [Ad& 0.3 mL ofa working bacterial citre om I6hhto 24 hin MIT broth (see 5.4.52), AGO. ml-of SU mmol L-CaCl 2 soltion (see $2.23, rill and incubate or 10 room temperate to allow asorpson of phages on ost acter el [AGE 255 ml to 3 mL of MI7 Overlay Agar (ste $22.3), peviousy superooted ina boiling waterbath or riezowave oven then coled ina 47° C wale bath Mix bly, thon uniformly srced he sarmples in plates containing MIT agar (s02 522.32). To obiana homogeneous distribution of samples he bors mst be tilted slowly by hand before soldfction ofthe ovtay age. (Cae must be tk to peeven an uncontled oveeeatng of the oven toe bling excessive and unsontlledliqutid aga. Unscrew te caps of te container to prevent ny sk of explonon {© Preparation of phages the interfering substance From fetes bacteriophage suspension, ad aid whey or ki miko obtain 8 ution of he later t 2m. The fal phage ste should he Between 8.10 and 3.1019 PRU Store his sugpension in the waterbath at (20+ 1) * Cand use witha 2 hows ofits peoparson For caumeratio it acid wey or skin mil, procced inthe same mans asin $45 ao Standards online the 27/01/2015 at 11:20 NP 172.281; 2014-11 Pagesspitit EABAT NETT281 30. SS Procedure for evaluating the activity of the process S51 Procedure for evaluating the activity of the proces according othe conditions of us transmitted by the manufacturer (bacteria, yeas, mols, spores, scutes, bacteriophages and mycobacteria) S511 Experimental conditions (mandatory adional mandatory and adiltonal Jn addon tothe mando conditions additional and addtional experimen cndions maybe hose cecoeing tthe intended se ofthe process, ame!y ntpsciransiate.googleusercontert.comitransiate_t 311632ier2019 NFT72-281 2) Duration of comact (exposure tothe dsaestion process: tae drain of contact may vary acording to the mabufctuters intrusions; ne contols ofthe survival of encin germs onthe convols Tay nat ge ress i. In thes cicurtancs, it wil not be posible to determine redueson rates logurdunie> 3, 405 aga equed bythe docuncat (004. 1) Teopeate mandatory tempecatue: 20-+2) © atthe beginning ofthe rest - adional compulsory temperatures (nthe ease whete the cuen: use i outside the conditions lemperatres above) fr large volumes: 15-25 °a the besoning of hee any other mperatre according othe regucemonts of the diferent sectors of actvisy. The aniieton for choosing ‘hi smpertue mus be provided in the epor, under she responsPlty ofthe manufacture ©) Moisture + Moistze equceaent 40% o #0% Relative ui, oly for asus where eels humidity are encountered sd nthe absene of specific resommendatons at Fst ty a oma mandatory hari inthe case where the ctrent wos guide the conions above humidity: Any oer humid acordng tothe requirements ofthe deen sectors oF activity ‘The jstification fo the choice of this amity ms be rode i the inate, unde he esponsibility fhe indi, volume oF he 00 “The process manuficturer spesfesthe lis of use ofits process in ems of te volume tobe tated. On his Basically, it wil be veri tht the volume of he test room adapted ta he proces nthe est a be implemented As for temperature and humdi, tsa seqised that the test he eepeated at vlumes citersst -compoleoy vole: 50319 150235 ifthe process elms tse woes ous the mandatory voles above test complamentry must be realized: ifthe poets lain only volumes of we outside the mandatory volume ave, ‘only the tert coesponng othe condtons of tse must be cried ot an obligatory, por Standards online the 27/0/2015 at 11:20 NF 172.281: 2016-11 Pagessgiitat LABAr 3 NPT 281 ~The bie below describes implementation method according othe case eneuntred ntpsciransiate.googleusercontert.comitransiate_t 321632ier2019 NFT72-281 Earn tae Sa toscana mc a trent tpg seen siereoee stg =e sminttmas eee nnn enced i ee end ich ot ng ad NEN ad eet sy {puesta na rin nn ce? aS art gs pce i a i ee beep pega amd hora aga ie ey at na Wl a we me ce We eae ual Standards online the 27/01/2018 at 11:20 NE T72-281; 2014-11 ABAT" i 3 ©) maure of med, orenation and locaton mandatory suppor stainless tel dies ~ aalonal suppor: any nonporous spot provided ta itis seibed ia the test epo 1) Cases of nonazeted devices ntpsciransiate.googleusercontert.comitransiate_t 334632ier2019 NFT72-281 + eriestation and mandatory locaton: distance as por Anion Bata eight of | ‘nd 15, vical poston, facie tothe oppose side ofthe apparatus + ericson and nitions Ioatons: any possibly depending on he intended use of te process, 2) Cate of directed devices (96 55.14.22). 1) Naae of ironganims obligatory mlcroorgnisms: 6.521 ~adonal miro-rganisms: any micro-organism depending onthe intended use of he proces 2) Imerferngsubsances For some specific applications, 2nd according othe method manuictrers recommendations unin inthe presence of other inerfering substance ae lio posible unde adiorl conto. S52 Survival indicators of germs om supports Te be performed atthe ane ie tthe atu! te S521 Arica contamination and dying of supports CContaminst he media (52.3.1) placed in ster Ptr dishes by depositing 0.08 mL = 50 of suspension. For baceraphapes, deposit 08 mL. = SD ul ft baclephae suspesio} ‘Then homogenously spread the inoculum on the apport covering an area.’ m2 f05em2). ‘To otis, usa sil inoculation loop and perform the spreading by making several it ‘wordt cover the cect race (Ie sprening deposit rt cover an area approximately 2 min dare) ris imperative to change the hanle beeen ech spr For cach ain, contaminate wo dental copes of ach ssp. ‘Thea dy the suppons ‘Toe dying ofthe suppons canbe cased on aecording to different methods (PSM, ove vented rat, desiecator, beach, ee). The spp ae place in Bel sh id ope, ten sabected orn for ‘maximum durston of 120 min at termperane ot exeeing 37°C (samples not being dy) heen of thi prod ean ot be wed fortes) 1848 upto she laboratory in charge ofthe tes wo regularly monitor the tat ofthe depois 9 a8 nao subjocto drying conditions uaneessrly- Once Vshly ry, he suppor ae removed and ie Ii ofthe Pett dish scored [NOTE | Refre sbjeing the abseesto eying itis porto conser: allowing pons “Tae scsi descaton of the eifeen tet coors may ay spicy tg fr onal res afer dying ad expe are Bawa st th semary thle ie Aaa. NOTE2 twas obuvedietets a low epee (28 1)° Cand am dying aes rua for seve gms “pag paeantiane ™ 7mransan20 NP 172.281: 2014-11 3 NETTIE (Once the drying is completed the period of exposure to the dninfeton proces begin (pei more o es long tem defined by the manufactaes to btn the inactivation of microorganism. The contl support wil be ot exposed the disinfectant and kept in the laboratory forthe durton ofthe contact time. A the end ofthe petod the numberof the wo suppor (see 58.122), ntpsciransiate.googleusercontert.comitransiate_t 340632ier2019 NFT72-281 NOTH} Krowing that he inti it snd on col madi andthe int of onal iemaganis on eae of Tbocus (7 os) ma Eaves impart on the sod oan of compan i ee Scam ‘erequ, arson proceso dj he lal cues oct above he ett bye send Siy, he yng echo at be excl eleste, epsilon thir sexsi to dyin, [NOTE nthe cse ote ered out on he same process a leading 1 conlsions of eonteriy W ‘sop cen, eps oe tei eons wih a yang end coergening he es Ahelowest anda ie sume eames vale, ‘Whichever drying mito is chosen shouldbe documented the et eprt and allow obtaining» microbial ire and a quai ofnerfring material in accordance with he vera ofthe dosume: 551.22 Emumeratonofmiroorganions on supports Atthe cud ofthe dying tins the wo supports ate eft in dhe lose boxes ding the time provided forte :et (exposure pero) under contons of temperature abd relative hit close fo those measred nthe oom where the atl disinfection takes place Atihe end ofthe exposure period, ane tbe two media soe inthe labora to ‘0 conial contains containing 100 mt of stele saline (52.2.1). Shake manny fora ew seconds. Wi gle shaker or tsing the tp of Piet. Scrape for east min inal diestion the surface ofthe suppl o deacons, Scraping maybe supplemented by he use ofan ulrasonie ibaor for 1 min spocly ‘equerey ae power ofthe vbr used and look fo a possible ambi eet before se, the ‘ator i ted for he tot, imu als be for the witnesses under the ease conto. Af ues api gittion dite Lm of tae recovery Hid in 9m of 5.2.2.1. dient ad continue to cmy out succesive ditions yp dion 10. (dition tobe adjusted according othe prepared auspenton) Tanfering twice Ira ofthe recovering Hild tthe dao 102 and 10. oblaned hove in Pet dishes for couting by inclusion in the agar medium $221.3 for sans $21.1 1052.14, by inlision inthe 5.2.2.6 ag medium for strns 521 and 5.2.1.7 by the §2.2..11 agar mci fr sins $2.15 and inchsion iaager medium $221.10 forse 5.2.1 13 0 5.2.1.16 ination For each tin, count ie colonies inthe agar medium, determine the averages forthe dilations shove mentioned an rept the retl the number of bacterial, sald spores or pores acter recvered a the supports ie. [NOTE Telos of viii (ying expo san be cle ad mesond inthe erp N-T 551.23 Emmeraton of bacteriophages on suppote Atte end ofthe dying time, the wo supports ae eft inthe laboctory i heir lose owes ng theme sheds forthe test (exposure period) under contons of terpenes hygromry relive ta those measured inthe room whee the actual disinfection ake place. Attbe cud ofthe exposure pvid,uansfer the two media sored inthe arson to ‘80 conical contains containing 100 ml of ster sline (52.2.1). Shake manually fora ow seconds. With gla shaker or tsing the tp of ict. Sorape fra eat 1 mini allicetions the sure ofthe suppnt to detach bata se NOTE Theaseafaneeaocevibstr ponte desnet fh pags om spp TOE Standands ovine the 27/01/2015 at 11:20 NP 172-281: 2016-1. ygeRRE LABAT NET M Aer fre rapid rng, die Im ofthe recovery gp in 9m of Phage Dien (600 $22.4) and continu teary out successive daons upto dition 10 5 (ilaion wo be ties oF suspensions prepared ntpsciransiate.googleusercontert.comitransiate_t 351632ier2019 NFT72-281 ‘Take duplicate 0.1m ofthe dutons 10 nd 10 aa asferring cach spl now separate est tbe, [Ad80.5 mi of 16-10 24hour working bacteria ule to MIT broth (5.45.2) ‘Add 0.1 mf $0 mol/L CaCl slain (se 5.2.2.6), ell mix and incubate for 10min, _mbentenpersture to allow adsorption of phages on host bateris sll, ‘Ada 2.5 mL. to 3m of M17 Overlay Agar (62 522.3.) previously spetooted in oiling water ath and then cooled ns water ba (65+ 1)° C. Mix rie hen spread evenly ‘nples on MI? ug plates ee $22.32. Toobin a homogeneous disbuton of spe, hand boxes shoul be ltd slowly before sliitication ofthe overlay apa. For ech amin, coon th number of phage inthe ag mst, determine th avernges forthe stove dltons and eae the result to the suber of phages recovered oa he supers, ie. SS13— Preliminary validation tes for the abvence of residual effect SSI31 Recovery of dafetan resides onthe surf ofthe supports and research of her possible eet antinicrobil Make at eat wo suport: a dtnce d defined inthe tube in Anex B, inceutun in vere! position vented in he deetion ‘oppose othe disinfection apparatus for automatic processes on which the direted pry wl be sprayed forthe directed says. Media preparation ile wih preston pipet 0.05 m= 50 Lofecontied milk edt 1/20 or subtance ‘interes only died 1/10 w/in divest $22.5, For Sgile gums as deine in 3.5 drop SOL of reconstituted milk 1/5 for esting retaining this option Allow the suport dry win 58.121 acting the media withthe producto be tested Expose the supports othe produ dispersed inthe test room or pray the py ected thelr surface, to obtain the same dsnfectanFation a ding ats. Recovery of deposited disinfectant Remove the mein arte manufacture’ lames contac time and ane to L00 malo gut recover. [Let be thi cotton oto, S8132 vestigation ofan inbory eet nagar na Ptr dh, place I lof the dilation 104 nor 10 an 10-4 and oe 10-4 and or 10-808 suspensions of microngtaisna (depending on the ype of germ tte, se $4.3) and 1 eof staon S obtained | 55.1. without binging the two lcuds at conte. Fo bacteriophages, sete same method of count in 5.51.28 (or he eleva lations Pour the agar medium acconling othe san, mix wel coal ei neubate Compa the mnber of colonics obtained otha found by non-contact agar eatin with hit Solution for olestng disinfectant esidues NL ‘pagar 7020150 1-20 NP 72.281; 204-11 -35 NET R281 $5133 Investigation ofan inhibitory eet on membrane fers ntpsciransiate.googleusercontert.comitransiate_t 360632ier2019 NFT72-281 Fille 98m of slton $ obtained in 5.12.2 on membrane and inset times wih SO mL of gud (5221.8) {oman te produc, Cover he membrane fourth time with $0 mf ofiguid (22.1 8) and add 1 mi ofthe suspension diluted to 10-4, 10-5, 10-6, 10-7 andor 10 Filler and bring the mershrane fo he age men fo counting Perfor his et np an proceed in the exe way for ach of he el ri ‘After incubation, cant the colonies found ed record the average result ofthe two tess perfor. Compare N2 Se munber of colonies obined by Hization of Im, died sutpenions 10-4, 10-5, 10-6, 10-7 ‘andor 10s on membranes tht have not been in coat wit the divine, ‘The fiationteshniqe is ot applicable Fr bacteriophages and wil othe performed 55134 Ivetgaton ofan inhibitory fet due othe support the aur medion na Peat dish deposit Lm ofthe 10 dition 10-+ 10-4 10-7 andar 10 microbial suspensions tnd the exposed support obtained in 5.5.122. Pour the agar medium according a he stain, mix wel coo an inet. Compare th a3 sumer of oon obtained fo ta found hy agar count without the addon of NE support For bacteriophages Pave te supports on MID agar base Mixin one eb 0.1 mL ofthe affected betriophage ditions wit 0.3m ofa beter cular ‘working Hie rom 16h o 26 a MIT bot and 11m of 50 mmo L CaCl. soliton mix Iwiefly and incubate fo 10 nn atcoom teers. Add 2S ml 103 mi of MIT Agar Agee, previosly supercool in boiling water bath or microwave ove and then cooled ina waterbath at (48. 1)°C. Mix bey and thon spread the samples evenly ove the agar lates background MI7 conning the supports, S514 wayne S841 Anica contamination of media Proced asin $..1.2.1 and prepare hee supports per, S542 Conacng the media withthe product to be tested S5:1421 Cas of nonsiteted process ‘The mandatory conditions ate bopining of the est at as flows: temperature ofthe test:oom -(20+2)* Cat the begining ofthe est ner the suppt as emperatze reference lative humidity between 0% and 0% the Benning ofthe ts: each torque Const ime concentration climed by the mor fairer sal ets "The system for obtaining the our temperature and hypromety condition must be topped daring the daraton ofthe et to avoid any parstc bance ofthe ai ‘The est s00m, stale, and thesmally insulated, with abomogeacous epeatr and ative humidity, rast beswsen 30 m Sand 15073 As proviusly dsered (5.511), “emplementary* mandatory conditions may be fulfilled fr processes whate use would ley be ouside the mandatory conditions cove. ‘Cute ouetsto prove eaage of aso ipod product, Berwcen cach test, remove, by any suitable means venilaon-etacton is mecessary), any residues oF prot that may have resi aston nthe nex test, Fab Standands online the 27/01/2015 at 11-20 NP 172-281: 2014-10 Paga"3BiFRRE LABAT NET 236 ntpsciransiate.googleusercontert.comitransiate_t 371632ier2019 NFT72-281 nde the they suppor by sin eighth) between | m= 1S mata dtance (defined Gran ce ca aac o Poston af the supper: veal (riematon: Microbial inoculum aimed in the opposite direction othe disinfection device Start the process ad pred the product or the recommended time scoring tthe volume ofthe room, Determine revscly the qunttes fused by weighing or volumetric measurements ‘Remove the modi arte exposure time laine bythe manufac, ‘Te rom shold he veiled afer each tet an the tet mia shoe anfere immediately ster he est. the ecoery iui Provide a sale test diagram, indicating the exact cottous ofthe oom, the position of the Aisinfston a wel as gor ati foreach manipulation performed ‘The paculr case of metre UV is described in Appendix NOTE Themathd esrb hare erste ot cans considered to emia in ner ele to slam einstein acoding oh avument i aera pee the eecswenet of hese UV 5514.22 Case of diated processes Non presute processes: ‘The dee supports (donot use coneve suppor) shuld beheld borizoually, vente inoculum upwards, stance of at east 20 em exept for rated UV proceises fr which i distance mus corespond othe maxinum ditanoespcified bythe manufactur. ‘Any cus must be prossibed because takes the est ulaerpcabe All paramecers that can in uence the atv ofthe process must be peed: media position, time evsson of the proc, cont tims average mas ofthe dpe prot iin on the sop, spe craporstion ane distance device supp, Processes with peter: ‘The thee supports mast be belé vertical}, inoculum dccted towards the manipulator, aa distance to he defied by the manuiietirer, Any run mathe prosaibed because tries the et erimerpretale ‘These procenss ae therfore ikely to lead dplcerent of misreopainms ou ofthe srice of the inoculum, the text method sould allow aol or reprsetative recovery ofthe gms, All paremesers that can in uence the actviy ofthe process must be specie mea postin, time prot emission, contact ime or sect of rave ver the et are, average mast Aspersed prot onthe sustae, evaporation ate und distance hrs the mei ‘The codons of plication must nperavely be communicated by the manfatue for bach types sor 55143 Emmeration of bocera, mycobacteria, yeasts, mals spores bacteria and bacteriophage recovered ‘Transfer each spon ax soon ait seminal fom the Les room or a he ead of the amie ime with the Directed ispersar, in 2 container 10 ml of ster saline ($221.8, ‘Shake manually fra ew seonds. Wit asteie lae shaker or sing th tip Piet, scrape the suie ofthe wel dit fora leat min nll dietons a deach he tmiroonanians, Ienccesenry, ute a ulasoie vitor (5.3.2.2. Aes fre aid again, dilute I sn of the rosovery liquid in 9. of 522.15 luca and coatinue to make successive dations upto diton 10.3 cepa nan a0 NP 172-281: 2014-11 pagerssikits “37 NPT 72281 ntpsciransiate.googleusercontert.comitransiate_t 381632ier2019 NFT72-281 Taser | mL ofthe pure rsovery Hid ano its tions 10-1 10.2and or 10. twice 2 faetion of the presumed efficiency ofthe proces in Pt ses for enumeration by inesion in he mei suitabe apa (5.2.2.3,5.22.1. and 22.1.1) For bacteriophages, use he sae method of caumerton a ia 5.5.23, Pour oa oae bad 10 ra of the covery lid witout doppie thesuppst iva ster Seaton appara the separately in another tering aparas the remainder of te lig (tou: 87 mL}. (Ihe Slaton eehgue is nat applicable for bacteriophages and therefore wil ot be rot alized) File, wah the membre thw ines wih 0 sa of salvage ui (522.18) ba wl have ben befrehand and sired inthe conical ask withthe suppor. ‘Then afer the membrane tthe agar media for counting (5.2.21.3), (62.2.1. (52.21.10 (6.2.2.1), said na $m thick yer Aseptcly remove enh medium and place np dshnoctrn up.Sik a sulfiietamoun of agar to cover the suppor homogeneously. For buctrihages: Place the supponts om MIT agar hase. Mix ia 0.3 mabe oF a ceria ule working fom 16 bo 24h MIT broth cure and 0.1 mi of aC» olson 50 mel L,befly mix an neue for 10min at oom erpratue, Add 2S rato el of MIT, previously spereole in biling water bath o microwave oven ther cooled ins waterbath at (47 1) °C. Mix ely then prea the samples evenly the M17 aga les coming he supposts Do the cane for each ofthe contact an atest sina foreach of he suppor SSL44 Incubation and enumeration ofthe et mire Incubate: 248 ALT" C oF36° C= 1 for baci song (21.1 1052 1.45 = 72930" C 1 with eaing at 8b for spores (5.2.15): 88 at30°C #1 far. aame (5.2.1.6 72 at30° CL with 48 beading oe A, Brain (8.2.1.7); hor 16-24 hat 30° forhacteriophaes (5.2L. 11 and 82.12}, = 21 daye 35° Con3T*C# 1 °C ipa ine CO 2 ncubator) for myesbacterl eine (2.1.13 nd 52.114), For cach amin and each typeof support, coun the olonie that appeared fom the icons inthe Pats ise nd on he membres. Calelate the result (average ofthe thre test media eorerponding to the average number of bacteria, citer he average number of yeast /anlds othe average numberof viable spores found in 100 recovery iui) In the sme way, count the colonies appearing onthe supports nla in the agar medium, ent he average coesponding af the dee tet 552 Viruldia teste ‘The experimental eondtons (mandatory and atonal are described in 5.5.1. 5521 Oveniow Before peeforming atest equilibrate al agents - withthe exception ofthe et vrs suspension = ie the itrerngsabstnes, was, ll eal mea, dunt and test salation a the product s1(20=3) Cor (10+ 2)*C using a thermostatic bah ‘The es at ld with cecold medium (MEM 2% SFV) fr he vais titans to be perfomned eer stern conte anes nd placed ina hth of mebing ie. Mica pstx are labeled for ietifeton Afro: Standards online the 27/01/2015 at 11:20 NP 172281: 2016-11 Pagevattkt LABAT NET T2281 23 ntpsciransiate.googleusercontert.comitransiate_t 391632ier2019 NFT72-281 5522 Preliminary st to valldate the absence of residual effect 55221 Recovery of snfectant residues onthe suppor and evaluation of he potential vince fet 55222 Tacatleantoo mppors Located at distance ds efned in Annex B, with» vera incutum aint inthe direction oppose othe disinfection proces. On which he dispersed product wl edict for manual process, Medi preparation Pipete 50 ofthe ue conning he recone ilerfrng substance wing precision ptt he appropriate fnlconeeration on se suppor and then sprang and hen dying (5.5.12. “Exposure of the supports othe tested process Epose the suppor othe automatic snetion process inthe fest room or manual process when the resting phase, Recovery of sopports and mentale ofthe atv of the disinfectant deposited Abe end ofthe conte time, recover th media and nee ito 20a of recovery liqudt to neutlize the ation of he dsinfotant on he sce, ‘This gives the ecoveryslutionS NOTE Dion neuron oration pci agent hd end capctin he reget. A Incomplerewralision of fects wil vat tet, 55223 Sent of els tthe vias ‘The reduction of sensitivity of lt vrei ovals by comparative trations ofthe vrs on cells trated or nat with disinfectants Call eaens 2) eal mat: 0.1 ml. slain Sor PBS is dispeso iso each wel the microplate £96 well. Aer [hat 37°C, the supematan i discard cel in mapeion: 1 rl of solution Sof PBS eae oa volume of ell suspension concentatdtwie, After | hour at 37°C, the cells are ceniuged and resuspended inthe medium, fale Comparative ration of viruses: The vrai 102 to 10-10and vated on ete untested cell in parallel. There ian absence of Jos of sensitivity when tis soon S 2) indicts alow degre of cll destruction (25% of el in mouolaye oF by produces eduction ofthe vin tite 1 ag 55224 Contol ofthe efctiveneesof he cession of difectant act A volume of solution Sor volume of disilled water i aed to volume of vi soponson mixed with fhe letersi substance. Ae 30min cost a 20° C iin series ae performed end he Gers is re soraaze, ‘The ference between he ils mat os than 0. Tog i Standards online the 27/01/2015 at 11:20 281; 2014-11 PagevafitRRELABAT ntpsciransiate.googleusercontert.comitransiate_t 40632ier2019 NFT72-281 = NETT228L 5523 Testielf $8231 Anica contamination of media Prepare three supports per srs [ine volumes of vrs suspension ate mixed wi 1 volume ofitererag sible or each et prepare tire ts supp an wo est sports by deposi 0 il fhe suspension deste above. Then proceed othe peading and dying ofthe supports as previously dined 65120) Use dey me fo the next hor to aid inastivation of the is de to went, 55232 Espane of the supports 1 the etd process 55233 Determination of viral tire afer exposure Immediately afer recovery, a the end ofthe contact time, cach aver must beansered 0 vial containing 20 mi. of eevery Mid The amount a ecovery iid can be raced long ils ‘Neualzing power rensnscompian NOTE Hsrecommende tht sing bata fom peli ets eset he Toenestalntin ofthe eiues Barbee ope Mix by hand fora fe seconds Using sterile gas shaker, scrape cach side of ie to pck pal races of dried eidue. is also posible to use the vibrations beasonie (83227, Aer ial aid sting dice t mf reovery solution in 9m. and continue the dilations in sein. The val tires determined nooring to 5.44, S524 Calculation and expression of resus Protaca of es Cell culture ets ce recoded 8°" for lak of PCF, "1" (approximately 25% of cls with ECP) 1°" (all ells with ECP) (ECP tiaton) or 1s many ys ngs (rng forming is, PP), ‘The estimated concentration af the vin sell be calelated using appropriate methods: ‘The caleultion of the vil onsentrton cane cri ut vs the Spsrman-Krber method (eee NFEN 14476 and NF EN 14675); ~caleultion of PFUs: the aumber of unis forming a range (PFU 1a) mst be calculated. Only crop units ‘ell with noe-convet ysis anges should be used forte eaeuation Redston of viral teri clelste hy dierentitng between viral vs dried before nd ster eaueot by the dsinfetion proces Verifistio ofthe methodology testis only valid if the following vteria ate met 2) the teat ia suspension has 9 TCIDS0 of hetween 1107 / mand 1105 /m, of haa east one tite ‘whic makes i posible to determin a redton of og ofthe via tine; the detect ie redueson i east 4 og «sonra ofthe celular suscep: rdution ofthe vi tne V)) > 100 where V= 0. in the case of phages counted on spp S65 Determination of values obtained i the preliminary tet (651.3) Search fran inhibitory eet in agar: average values obtained + Search fran nkbitory eet on membranes: verge the 2 vies obtained, + Search fran inhibitory eet dae othe agar medium: average the 3 vals obiane, 566 Determination ofthe umber of germs and ranges on comiact-est media and rate log eduction rato (68.143) Fortes main represen the mer of rising gems (or plages) in 100 ml of Aid recovery is bined frm he isan 87 af ecovery gid enki he ule of 9), the ceameration ona memvane is tetera 165 for basta and yest or 85 for mals, reson he mambor a> 165 (155), trom the Hatin of 1 mi ofecoery iid from the number of colonies or plaques obtained from 1 mi ofecovery igi, a tion recovery id ‘Or from we sucssive ution if the weight alvalstion is possible ‘The number 2s the numberof colonies (or anges) obiaived diet by inchsion ofthe test medium tis ceumsrston ons boi gsr han 390 for bactia nd yeu o 165 for molds andthe phges, recor the mamber a 350 (r> 165), For phages ote 165 PFU, ‘The sum + 92 eorerpond othe total number of surviving pms (or phage) per et oppor ‘The log redution ate is abs by taking nt acount the number feeds (er phages) othe witnesses , onthe other hand the numberof surviving germs (or pages) +22 ntpsciransiate.googleusercontert.comitransiate_t as632ier2019 NFT72-281 Pde Standands online the 27/01/2018 at 11:20 raigeRRE LAAT NETT2281 “0 “The formal to oblin is vite: dog Flog dal 4 92)=log (7461 +931 ‘Once the log reduton obtained for each rest medium, the average oF he hee media willbe elzd, ‘To cnsier haa decontamination proces i compliant ts mpertve ha the average ceucton logarithmic is greater han or equal tothe requirements ised in Table ‘counts eading to calculate ann? areal equal 0, thn the reading of log. er atest wih € alioan anda somtolT 45 108 gems pe The survivin gems counted ae =22 colonies on average for Im of pure recovery Mud 15 colonies on average For ncuson of the agar mei. n= 22 100~22 10 reeds 100 mb pans, So, the esl Flog TH (at 029) log 45.106/@.2.103 +189] ord 3.3, [NOTE Inthecas wher one ofthe uc andor 2s indsteminale (> at ne fh mis metiondin 61, activity tthe vale calelted nog fo hs it EXAMPLE? Number felis 30 and eos est ras, ‘The surviving gems counted are (othe thee suppor): > 880 colonies for | mE of he 10 atin ofthe recovery au > 380 colonies fr istsion ofthe aga sedium, So 11> 350210 100, fe 3.3.10 snd 100 ms o> 30, We hereire haved =log | (81 2)] log (45.106/(@ 3.105 ¢ 330) ed. og, EXAMPLE Low munber of clk nd ite fret of eg. erate wth Coton anda onal T=. permet ‘The srviving genes counted are (ore nce spo): 3 colonies 87m of fitered recover Haus 0 colonies 10 lo ered recovery liquid, 0 colores nm of pur ligudeecoery and ited 10 ered 0 colonies for inclusion ofthe par medium. so wt mo, We therefore obisin d= log 7 ~n2)]= fog 5.10 (3+ 0). e607 lg ntpsciransiate.googleusercontert.comitransiate_t NB 972.281: 2016-11 a32ier2019 NFT72-281 Tg Standards online the 27/01/2015 at 11:20 NP 72.281: 2014-11 (ERE LABAT 2 NeTIN28 5.7 Results interpretation damental values tle of wor aspensions N most be understood: between 5.107 and 2.104 CFU/ml. for bacterial esas rin 52.1.1 9 2.14), Pocentilly 3.109 CFU mi for Parudomonasaerapinaa (ie 82.24.11 and S20, ‘tween 2.107 and 1.10 CFU/ml. or Candida acne (nn 52.1.6) ‘between 5.10 ad 1.107 CFU. for Asperla Braent (ran 2.1.7 ‘between 2.10 sand 5.105 CFU/ml forth pore-Foming in of Baila subi (rain 52.15 petween 1.10 Ya 1.109 PFU snl (TCID so) for vis sans (52.8 8. etwoon 8.10 rand 3.10» PFU mi for bacteriophages (6.2.1.1 and 5.21.12) ‘between 1.10 ad 1.108 CFU Fol for nyeobactrl seas ssi $2.13 and 821.18), edi conto ot exposed to disinfectant: oral strain tented T mut be | lg hove log reduson objestivs as dfinod in Amex D; = prliinry tents (ae $5.9. Hot 05 Nt mE O5NI nossa ‘The experimental conditions are applicable during the txt fr the quantities of prt used: Hl 5 noconding tothe ids of aplication (4125 according tthe lds of pplication ‘+ Minimum rate of reduction on bacteria spore: spricidal activity: 23 + Minimum reduction on yeasts and molds: fangidal acti asad mn yeasts yeast acti ntpsciransiate.googleusercontert.comitransiate_t 45163,2ier2019 NFT72-281 aona Td Standards online the 27/01/2015 a 1-20 gree vAnAr NET R281 “4 ‘+ Minimum reduction rate on virucidal activity againet bacteriophages an ene ‘+ Minimum reduction rate on myesbacterias mycohactericidal and or tuberulacdal atv: asad spending the nnd ue of the proces, it may be necessary a Asnfectnt activity dx 3,4 or Tog depending o he typeof acoorgaiss ested: bata spores (© 3 og) yeas mold #Tog-ectria 5 log)- vrs 4 log) - mycobectria 4 og on various stains adapted othe sectors of ws S58 Test report EXAMPLE OF TEST MINUTES -Ieentcton ofthe est nortan sample identfiaton: proces tame (product and device), devise pe batch nd sei mamber (fasows), manucactrr, dae of cei, stage contns, active substances (optional), dion of newralizos nthe media or dient, nate of e fering membranes, lbasnic water; est stat ate and end dat of tet, limits of ese in particular as regards eapaciy of isin nthe premises (minimum volumes nd maxima). NON-DIRECTED PROCESSES tet room: vhs and dinensions (ight, width, depth) tempera and relative ham in bepaning and end of the tes, daseriptiog, total rea’ volume rs; [NOTE | Moire an tengertr nat es tram ia canons atthe nd fhe tes ~quanies of roduc uted ison ime, nial quansiy wae in the cate fa proces of 19 15009 Tempers 15 C9 25° Cath eon af he tet Acorn the ned TRO Standards online the 27/01/2015 at 11:20 NP 172-281: 2014-10 JERRELABAT NET ost ‘Appendix E (Informative) Specific methodology related to the evaluation the antimicrobial efficacy of the processes of non-directed ultraviolet radiation disinfection E1 Overview ‘The method derbod in this annex i pecially concerned wit he evaluation of the ntnisrobilefSeacy of rnoniroved ulin raison dsin‘etion mets according o his dosent ‘Urine ition processes ae characterized inter alis, bythe intensity ofthe wavelet ofradation thi eters exresod in Watl/ ex: >and the exposure dose (easy * tine) eammealy expressed in m2 ‘The effectivenes of ulrvilet radiation dsnfection proceses depends on the distance beween media nd the sending souree Is lo nlunced bythe presence of dstcles tha can generate 2onet shade. The nature ofthe surfaces encountred can Tad the elton of UV radiation towards surfaces ot iret exposed ose sours of urs ration, 1 oder to etblh a reeesnttive elation ofthe ey, the est suppl wil therefore be poitioned n= Shadow zone cag a wall whose reflective properties es indieatd is Anise 3 of his Antex Although the effetveness of UV decontamination processes is dependent on the conditions of use specific test condition are defines to verify compliance with the requirments ofthe performance ofthis document 1 the contons of se recommended by the masufictrr die fm the condition scebed in this Annex, the manufacturer shal carr ot addtional ett demonstrate compliance withthe reyuirements ofthis document unde the ecommende conor of ke (conbtons ligntoysupplreas). The disinfection effiieney of UV docontarinton systems will ave obo howeves, avays be demonstrated acorn othe exaditeryconlitions cording tthe tess described below £2 Evaluation method ‘The tes: oom shal be defined for its humdi, temperate and dimensions in accordance with uidance provided in this dcument (55.1.1 and 551421), 2) Nature ofthe faces ofthe rare ntpsciransiate.googleusercontert.comitransiate_t 561632ier2019 NFT72-281 Special tention willbe paid othe reesvepropentes ofthe wall acing tho et supers (ee Figure E), The later must be covered witha lossy white pint (RGB color R= 100%, V~ 100%, B- 100%; MIN 01400; NCS 0300:N) wit ate fish [NOTE 1 fn wlth charters Sxl above, arenoabl pane asta he dinero sland epg the porte eondiione aybe , ‘Teen ofthe room smu othe made oF UV reflective maa (cmos, etl surfaces, #6) oe sronaly absorbent gs, wood, for, Standards online the 27/01/2018 at 11:20 NP 172-281: 2016-11 Paga’sirkRE LABAT NPT 2281 1 Posoning of th supports ‘Toe distanced btven the radiation source andthe test suppor depends on the volume ofthe oor sd mus comply with Annex B of hie document ‘The sports wil be prepared a inicted 58.1.2. and positioned vertically, wit the inocu orieted in conrastto he LV method, sidered for roncieted disparsion device (5.5.11 (). In order to materialize the obstaces Shadow zone) tat canbe found in room during its decontamination, 'tnon-raplucen and nor-porots panel (sinless te, HDPE, te) of dimensions 0.5m x 0. m wl be plat 2 fom the ground, near the tt supports betweea tem andthe source (Se Figure 1) NOTE? The backers un be dui atacel othe pal The dance btwesn the supports ad the pated wall (#2) mas be 0. m ad uinteaped. ‘igure H.1- Example of positioning in ales room (30 1310 150 m3) ‘during UV disinfection E3 Results interpretation 3.1 Atthe end ofthe disinfection the support are recovered and rested ak nde in $51.22, 6.5.5 246 nd 55.143. ntpsciransiate.googleusercontert.comitransiate_t 57163zierg019 Ne 172-281 Pa E31 The clelation rules are those mentioned in 5.6 ofthis dcamen The periarmnce requirements ate tote define in 5.7 of this document Fd Standards online the 27/01/2015 at 11:20 mgERRE LABAT Nero 256 ‘Appendix F (Normative) Procedure for evaluation mycobactericidal activity F.1 Materials and reagents Fd Test microorganisms Mycobacterial acvity nthe media nd industrial fields mst be east wing he sain teat Mycobacteriom terrae ATCC 15755 Mycocobactiidalacivt in the veterinary Held shouldbe evelted using the fowing stain yeobacerum avo ATCC 15769 =P] 05415, 1.2 Culture media and reagents 1.24 Middlebrook and Cohn medlum 7110 + 10% destrovealbumin complex oleic ald (OADO) (designated by THIVin the text) To perfor cous of isle mpootctei Giver Sls ater (G00 $221.2) ¢ 895 mL cat bolng to bisa complete istluin.Stiiz ia the auc for 10 minutes 1121 Candallow to cool 9 50° Co $5 Cina water bh {Ad 100 of Midlsbrook OADC Enrichment Mesum under aseptic comdtons, Sn pl 6.6 € 13 Apparatus hitps:ranslate,googleusercontent.comfranslate_f NE T72-281; 2016-11 581632ier2019 NFT72-281 13.1 High speed homogenizer, POTTER homogenizer F2 Preparation and enumeration of the test mycobacterial suspension in presence of interfering substances 21 Mycobacterial test suspension 2.1 Conservation of the test microorganism ‘The es coorgnit and heir pret cults shal be propared and preserved scordanoe with roquiremiens of NP EN 12353 Standards online the 27/01/2015 at 11:20 JERRELABAT 2.2 Working culture ofthe test microorganism In onerto prepares working culture of the test micoorenisn,prepere ft sbeultire fom the parent ele (F 21.2) Sak ina eas Wo plats of 7HIO medium (42 2.1) and incuba aa neabator 06: smovenar 35=1)" [Ae 21 days, simialy prepare «second sult fom the fst apd inate it 21 days "The fs ano second subcltre (9 consti (the working clr (0), Never ne or ate thi seule. In the case of aon test sans, any deviation fom his method of cuivationof| ‘mycobacteria or the preparation of tispersons should enol in he et opt indicating the reasons ht usted hse. 2.3 Preparation of est mycobacterial suspensions inthe presence of interfering material 1) Prepare n homogenous suspension adapted from the woking ela (2.1.2) by homogenization sng eas beads using a pase Handle, wtsfr test erorgaiems oft least, two boxes ofthe working erp (F212) ina wrew-cap and conicl-based tube consining 6107 g as ead (3.2.11) Test mierosorgaiams and 2 lof water (52.212) ae homogenized ‘by axing (5326) for at east Sino evenly diguout them on te beads and on most pars ofthe ane sure ofthe sere cap ube (53.222). Ads 10 mL of water (2.2.1.2) ops nd eesspond thor by mining thers (5.32.6) Ar Sedimentation 20 min, the superutan is rarsfera to a be; ‘homogenization wth the Pater type apparatus (F.3.1) using pipet, transfer SO mL of waer (5.22.12) on each ox (atleast Wo) ofthe work clue (F 21.2) Andeiove the ‘est mico-rgaisns using a iss sacl, Using e pipet, ioduce all the ligud bonos in 25 menting tube, Makeup to IN lof water (522.12, fora thse concave washes wih wae (52.2.1.2), (sifu ach te at aproximatly 2000 Nv for IS mints) After each enigation, remove the superatan,resupent ‘ining and iling with water the erga volume, [Alter tho last cenifagation, remove the superman and user the sez container ntpsciransiate.googleusercontert.comitransiate_t ) °C. in ade to preven descaton ofthe dishes we # CO» seubstr a place the dishes in polyetiylene bags or sealed with insulation tet peevent dying af the medium and then inebate ie NP 172-281: 2016-11 NeTT228 591632ier2019 NFT72-281 1S ma. lass ofthe Per apparatus (1.3.1). il with water up 11S ml ($22 1.2} Mix, col with ice and homogenize for 15 rin |e sedimenation of 20 inate ring which iti necesary to have enough ceo allow ootig, the supernatant is tansfered oa abo "Note: Other bomogenzuton sats ae allowed provided the amber of waits ‘colony forming pe liter obtained i appropiate and stable forthe duration of he tet and than exznation tmiroscope demons tha the suspension i as bomtogeneoi as fer the two serie methods shove 1b) Addo the superna he nefrng substances forthe disinfect se inthe rc domes acini. [Nine volumes of spematan mixed ith vole of imereing ssbstance Aint he mamber fells inte test suspension wth diluent (5 2.2.1.5), The make of cls canbe estimated by means of specuuphotomete ora nepeleneter “The inal ie ofthe test suspension must be between 10> 107 CFU Jen and 10> 108 CFU a Store this est suspension inthe waterbath ar (20+ 1) * Cat use the preparation in the ey Afar Standards anne the 27/01/2015 at 11:20 NP 172-281: 2014-10 Page'sbiikt'tABAT err 58 2.2 Enumeration and incubation ofthe test suspension inthe presence of materials interfering 1) Forte counting of the et surpesion made in paral he surface (1 and by fieation (2) Prepare 10-+ and 10- diliins of he tet suspension with diluent (52215). Mix 632.6) Sucice ‘Take sample of 1.0 mk of each dition in dupicme. Divide each sample ino v0 portions approximately equal size aad istibue thera on did dishes conning 1 lo 20 eof edi, ‘THLO on the surface (12.1, tris, a total of our boxes per 0m in duplicate iran: Coflcta 1.0 mL sample of sch duplicate dition an tans two args potions pproxinutely equa of cash surple fs # membrane faion apparatus (3.2.0, Filter and rns with SO mi of tins water (522.14 Deposit each mena (four ma) onthe suface of plates coating 18 mt 20m of THIO agar 12. 1 Trcukatin Incubate plats for 21 dat (36+ 1)° C212), Spread alle boxes os which ot possible ‘count the colonies, Count the boxes determine the mimber af olny fring nts (CEU) on sack bo, Incubate th dishes for an addtional 7 day, Dono recount thot where colonics are no longer present well separated way: Recut! the remaining Hox, Lhe numb is iasrease, remember oly the mst ih for subsequent calelations For cach bo, neste enaet numberof oie: (naga: note 330 whenever the number is pate thn 30 ‘a value of ss tha 14, record the number (but seplace With “14” fr subsequent calla). ‘On memrancs: Noe> 165, whenever ke aumber is rete tha. 330. a yaue offs thas 14, record the number (but eplace with 14" fr subsequent ales) Catelate he numberof Cm in est suspension NI and N2 using the metho indiaedin Article ‘6 ofthis document ntpsciransiate.googleusercontert.comitransiate_t 01632ier2019 NFT72-281 F3 Procedure for evaluating the mycobactericidal activity of the process 31 Survival indicators of sprouts on support ‘oe dn tthe smote othe ts. EL Anica comamination and dying of supports Scale wo scived supports place in Ptr dishes by depositing 0.05 m= 50 of he text suspension 2.13 (). Spread the icrovop and then dy (55.121. Afr drying the suppor ar kept in the Per dish curing the coma tie ofthe tein conten f temperate ad hind cove otha of he es Pi Standards online the 27/01/2015 a 1:20 AUERRE LABAT “59. 3.12 Enumeration of microorganisms om media tthe end ofthe contac me ofthe tes, nsfer each medi to 100 mL of reaver iid 2218) ‘Shake anully fora ow seconds and using sterile ls shaker sere each snfae of he supor to pickup the remaias of died suspensions Aer ial aid sting, diet ml ofthe solution in 9m of iuent (52.215) and cotinue seca ditions up wo 10 ilaton Collet 2 pf ailations 10-2 and 10.3 twice using the ecigue ofa surface coun (F223) Tneubaton of 7HIO plate ascording w F220), 32 Preliminary validation tet for the absence of residual effect 3.21 Recovery of sinfectanresiducs on the surface ofthe supports and ther posible search anicrobil effect Media contamination Inoculte to strived media placed in Ptr dies by depositing 005 mL.~ 50 i of dent containing the intetering substance ecostnted a the final concetration nthe test (5224.12, "Nine volumes of ie mined wi volume of iene substance Spread the mieroiop and then dy Contacting the media withthe product to be tested Expose the supports othe product during the same contact ine as during he esto obtain a ftasion dential sifetat onthe upon Recovery of the dsiafectant deposited ona support tthe end ofthe contact ime, str the mi to 100. of covery Hid (5:22.18) to resuspend she sinfetant aaced othe suppor. Let be the collin soliton ntpsciransiate.googleusercontert.comitransiate_t NE T72-281; 2016-11 NPT T2281 611632ier2019 NFT72-281 3.22 Investigation ofan aarinhibiting effect mated nl ake {ml of Callstion Solutio S, vide into wo approximately equal ied portions and inoculate agar pltes TH10 (F121). Leave dy, the plac o these same agus onthe surface I of te 10-s0r10-sation divided into equally sized porions using rice counting technique 229). For incubation and caution, oe F22 (2) ‘Compe the number al of colonies obtained with ths couning ofthe test suspension NL 2.3 Investigation ofan inhibitory effet om membrane fer noted 2 Filer ml of slsion S on membrane nd nse wih tre times 50m fied Cover the membrane with SO ml. of rise sind ld ml. ofthe 10-5 lation oF 10-6 laion ‘ter and taser the membrane to THO plats (00 F121, For incubation and eaumetaion, sos F22() and second the average of bth tests. Compare tN? the number of colonies obuied ror Standards online the 27/01/2015 at 11:20 NE T72-281; 2016-11 Pagesspintit LABar NET T281 0 3.24 Investigation ofan inhibitory effect om the carrie inthe agar medium noted nS na Petr ish ootainng 18 m0 20 mi of solid 7H10 agar (F121) remove the exposed suppor tothe produc, Place ml ofthe 10+ 10+ dilaton om the gar sure (2.2 (a) Compare o NI he ruber af olais obtied FA3 Essay itselt F334 Medio contamination Proced ain F 3.1.1 and prepare hee supports 3.3.2 Contacting mea with the product to Be tested F333 Enumeration of microorganisms on media [Atte end of econ me for dgpersal dete or when iia removed fom the est oom, asf eae support in 100 mL of ecoveryliguid (522.1. Shake manually fora few seconds and using a serie lass shaker, spe each since ofthe support pick up the remains of dried sutpensins Ate ia api string, dilute tm ofthe soon 9 ml of ene (5.2.21 8) and continue seca ditions up to 10.3 ation Transfering rice 1 milf pure recovering liquid and/or the dione 10.1, 10.2 and 10s by the acho of surface cout (22) Sncbaton af plac THLO FI 2.1), see 22) Filter he 97 i of the recovery laud Pour (Om fhe recovery guid into ane wit ‘vation ad the separately in another Fiering appara the remaining 87 ml of igi, Wash te tines vith SO ml of reaver igi (5221.8) tat ae Boen previously poured and sired nto the conical ask it the cane fr 87 mb). ntpsciransiate.googleusercontert.comitransiate_t 621632ier2019 NFT72-281 For enuneation an ition cunts, ee 2.2) “aks the support asopically and deposi she Pet di coming 18m 1 20 mo 7H10 agar solited (21.23) {tnt be the average number of viable colonies found in the 100 a. of esevery iid and donot verge umber of viable colonies found on suppor, F4 Calculations and interpretation of the results FAL Calculations: se Article 6 ofthis document FA2 Results terpretation Validated ent i ~The suspension tre of Mycobacterium avo NTCCS769 i erwcen 1.0107 CFU mL and 10% 108 CFU/ml ~The suspension te of Mocabacerum terrae ALCCSTSS i etWeen 1.0% 10 ‘and 1.0 108CFU/ mk; = Minimum te of the witness on medion tog 1 or Peliminary ete, a3> 0. NI and a> 0.5 N2 The reduction of the eis at eas 4 ag 8 ro Standard online the 2700/2018 at 11-20 Paga'eaiitkt: LABAT <6 Bibliography [I] NF-EN 1040, Antisnpace and chemical avinfctante- Quantitvesgpenion et for evaluation ast actereda activi of antiepisand chemical dnfctant Tet method aed prescription (phase I (lssifeation index: 72-152) [PL NF EN 1650 +, Anspice ad choi! dsnectams - Quanta suspension te for evatuaton of the fungicidal or yeast ‘the gro sector. in indy; the domes and community sectors Tet method and rouiemonte (phate 2, sep 1 (lasifetion dex 72-203) [B]NFEN 100881, Staines stele Part I: Lis of ails see (anificntion index Asssn-b vty of he ansepcs and chemical disinfection wed it NP 172.281: 2014-11 NPT T2281 SINF EN 100882, tifesesets- Part 2; Tena delivery condition for plates and sips made of corrosion resistant sve for general use lasifiaton inden: A 35457232) [5] NP-EN 18204, dniepice and chemical disinfectants - Quantitative sagpenion tet for evaluation mycobacterial act of amtsepies and chemical disinfectant sedi the fd ‘eterna Tet method and prescriptions (Phave 2, step 1 (lasfeaton index: T 72-802) IS) NF-EN 18563, Chemical dsinfotonts and antioptics- Quantitative til of germ carter for evaluation ‘he mocabactericidal or ereuocidal activity of chemical dinfctants ved for ‘human medicine - Test method and presrption (phase 2, step 2) (lassieation index: 772.286) 171 180.4793, Sitored labora itr - Porosity sale-Classfcation and designation ntpsciransiate.googleusercontert.comitransiate_t 63163