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Effective Date: 01.06.06 Sub ect

Revision No. :

SOP for isolation ! i"entification of Coli forms

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Effective Date: 01.06.06 Sub ect

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SOP for isolation ! i"entification of Coli forms

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Purpose +o ,efine proce,-re for #oli forms b! po-r plate met.o,. Scope +.is proce,-re is applicable to ,etermine #oli forms in a given sample. Responsibilities Microbiologist AM/0-alit! Definition : SOP( Stan,ar, operating proce,-re. Proce"ure /.' /., +est portion1 initial s-spension an, ,il-tions(/2%SO 63345 $noculation an" incubation:0 /.,.' +a6e t7o sterile Petri ,is.es. )sing a sterile pipette1 transfer to eac. ,is. 1 ml of test sample1 if t.e pro,-ct is li8-i, or 1 ml of t.e initial s-spension in case of ot.er pro,-cts. +a6e t7o ot.er sterile Petri ,is.es. )sing a fres. sterile pipette1 transfer to eac. ,is. 1 ml of t.e first ,ecimal ,il-tion 210/15 of t.e test sample1 if t.e pro,-ct is li8-i,1 or 1 ml of t.e first ,ecimal ,il-tion 210 /*5 of t.e initial s-spension in case of ot.er pro,-cts. Repeat t.e proce,-re 7it. t.e f-rt.er ,il-tions. Po-r abo-t 19 ml of t.e VRB$ me,i-m at 49:# ; 0.9:# into eac. Petri ,is.. +.e time elapsing bet7een t.e en, of t.e preparation of t.e initial s-spension an, t.e moment 7.en t.e me,i-m is po-re, into t.e ,is.es s.all not e<cee, 19 min-tes. #aref-ll! mi< t.e inoc-l-m 7it. t.e me,i-m an, allo7 t.e mi<t-re to soli,if!1 7it. t.e Petri ,is.es stan,ing on a cool .ori=ontal s-rface. Also prepare a control plate1 7it. 19 ml of t.e me,i-m for c.ec6ing its sterilit!. After completion of soli,ification po-r abo-t 4 ml of t.e VRB$ me,i-m1 at 49:# ; 0.9:#1 on to t.e s-rface of t.e inoc-late, me,i-m. Allo7 to soli,if!. Approve" )y Aut*ori+e" by Management Representative
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DOC. No. AV/MB/SOP/010 Page No. : Page > of 4 00

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Revision No. :

SOP for isolation ! i"entification of Coli forms

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%nvert t.e prepare, ,is.es an, inc-bate t.em in inc-bator set at >0:#1>9:# or >4:# for *4 .o-rs ; * .o-rs.

/.- Counting of t*e colonies After t.e specifie, perio, of inc-bation co-nt 1 -sing colon! co-nting e8-ipment1 t.e colonies in eac. ,is. containing not more t.an 190 colonies. E4pression of results:0 /.. 5et*o" of calculation /...' ?eneral case @ 'is.es containing bet7een 19/190 colonies /...'.' Retain ,is.es containing not more t.an 190 colonies at t7o consec-tive ,il-tions. %t is necessar! t.at one of t.ese ,is.es contains at least 19 c.aracteristic colonies. /...'., #alc-late t.e n-mber N of #oli forms per milliliter or per gram of pro,-ct1 ,epen,ing on t.e case1 -sing t.e follo7ing e8-ation(

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N 7 00000000000000000000 8n' 9 &.' n,: d A.ere is t.e s-m of colonies co-nte, on all t.e ,is.es retaine,B n' is t.e n-mber of ,is.es retaine, in t.e first ,il-tionsB n, is t.e n-mber of ,is.es retaine, in t.e secon, ,il-tionsB d is t.e ,il-tion factor correspon,ing to t.e first ,il-tion. Ro-n, t.e res-lt calc-late, to t7o significant fig-res. /...'.- +a6e as res-lt t.e n-mber of micro/organisms per milliliter or per gram of pro,-ct1 e<presse, as a n-mber bet7een 1.0 an, C.C m-ltiplie, b! 10< 7.ere < is t.e appropriate po7er of 10. /..., Prepare" )y Microbiologist &stimation of small n-mbers Approve" )y Management Representative
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Revision No. :

SOP for isolation ! i"entification of Coli forms /...,.' %f t.e t7o ,is.es1 correspon,ing to t.e test sample 2li8-i, pro,-cts5 or t.e initial s-spension 2ot.er pro,-cts51 contain less t.an 19 colonies1 calc-late t.e arit.metic mean m of t.e colonies co-nte, on bot. ,is.es. /...,., Report t.e res-lts as follo7s( / estimate, n-mber of #oli forms per milliliter( NE 7 m 8li;ui" pro"ucts5B / estimate, n-mber of #oli forms per gram( NE 7 m 4 d0' 2ot.er pro,-cts51 7.ere , is t.e ,il-tion factor of t.e initial s-spension. No c*aracteristic colonies /...-.' %f t.e t7o ,is.es correspon,ing to t.e test sample 2li8-i, pro,-cts5 or t.e initial s-spension2ot.er pro,-cts5 contain no colonies1 report t.e res-lts as follo7s( / less t.an 1 #oliform per milliliter2li8-i, pro,-cts5B / less t.an 1 < ,/1 #oliform per gram2ot.er pro,-cts51 7.ere , is t.e ,il-tion factor of t.e initial s-spension.

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Documentation : Microbiological +est Report References: %S 9401 2PAR+ 15(*00*

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