Department :Quality Assurance

DOC. No. : AV/MB/SOP/002 Page No. : Page 1 of 4 00

Effective Date:01.06.06 !u"#ect !OP for isolation $ i%entification of !&igella

evision No. :

E'(!(ON! evision ) 00 evise% Pages Description Original issue Date 01.06.06

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DOC. No. : AV/MB/SOP/002 Page No. : Page 2 of 4 00

Effective Date:01.06.06 !u"#ect !OP for isolation $ i%entification of !&igella

evision No. :

1.0 ,.0 -.0

Purpose )o *efine proce*+re for S,igella-isolation an* i*entification. !cope ),is proce*+re is applicable to isolate an* i*entif S,igella. esponsi"ilities Microbiologist AM-/+alit Definition : SOP' Stan*ar* operating proce*+re. Proce%ure for isolation /.1 ),e samples 0,ic, ,ave receive* *r ,eat treatment1 namel 1 *esiccation1 *e, *ration or po0*ering1 s,all be pre-enric,e* in a non-in,ibitor me*i+m s+c, as n+trient brot,. Ra0 foo*s an* finis,e* pro*+cts 0,ic, are s+specte* to be grossl contaminate* after processing s,o+l* not re2+ire pre-enric,ment. 3,ere pre-enric,ment is not re2+ire* 1 t,e sample is blen*e* in a sterile blen*er 4ar for 2 min+tes +sing appro5imatel 200 ml Selenite F brot, per appro5imatel 26 gm of one portion of t,e sample1 an* 200 ml )etrat,ionate brot, per appro5imatel 26 gm of anot,er portion of t,e sample. $nc+bate at 789" for 24 ,o+rs. From t,e inc+bate* sample as in 6.2 :inc+bate* at 789"; plate o+t loopf+ls on to &eo5 c,olate citrate agar me*i+m an* inc+bate for 24 ,o+rs at 789". $t is also a*visable to plate o+t on to t,e soli* me*i+m loopf+ls of eac, of t,e li2+i* me*i+m after blen*ing1 b+t before inc+bating t,e plate at 789" overnig,t as s+spect S,igella colonies ma be seen one *a earlier. $f t,ere is no s+spect gro0t, on t,e soli* me*i+m after 24 ,o+rs1 reinc+bate for a f+rt,er 24 ,o+rs. S,igella colonies on &eo5 c,olate citrate agar s,all be opa2+e1 0it, a gro+n* < glass appearance an* 0it, even margins.

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DOC. No. : AV/MB/SOP/002 Page No. : Page 7 of 4 00

Effective Date:01.06.06 !u"#ect !OP for isolation $ i%entification of !&igella

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0est for i%entification /./ Pic= o+t as man s+spect colonies as are possible :not less t,an 6; to investigate an* plate o+t into Mac"on=e agar plates an* inc+bate overnig,t at 789" to ma=e certain of p+rit of t,e colonies. P+re colonies are transferre* on to n+trient agar slopes to obtain a ,eav gro0t, for f+rt,er testing.

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/.1 *ioc&emical confirmation /.2 3ram4s stain ),e stain consists of a5 0.6 > met, l violet or cr stal violet in 0ater. "5 $o*ine sol+tion :1> io*ine an* 2 > potassi+m io*i*e in 0ater;. c5 "o+nter stain :0.1g ne+tral re*1 0.2ml of 1> acetic aci* an* 100 ml of 0ater;. 6.?.1 6.?.2 6.?.7 6.?.4 6.?.6 On a clean grease-free sli*e1 ver lig,t an* t,in smear covering a small area is ma*e *irectl from li2+i* c+lt+re an* in clean tap 0ater if from soli* me*ia. ),e smear is fi5e* b passing to an* fro over a flame an* coole*. "over t,e smear 0it, t,e stain :a; for 70 secon*s. Po+r off t,e stain an* 0as, 0it, :b; an* t,en cover 0it, :b; an* allo0 to remain for 70 secon*s. 3as, off 0it, et,anol +ntil t,e * e ceases to stream o+t. 3as, in r+nning tap 0ater an* appl :c; for abo+t one min+te. 3as, in tap 0ater an* *r for e5amination.

/.6 0est for motility 6.@.1 $noc+late b stabbing 0it, a straig,t 0ire into t,e top of t,e A+trient Agar me*i+m1 t,e stain to be teste*1 insi*e t,e glass t+bing into t,e *ept, of abo+t 6 mm. 6.@.2 $nc+bate at 78B" for 1? to 24 ,o+rs. Motile strains s,all be fo+n* to s,o0 gro0t, on t,e s+rface of t,e me*i+m o+tsi*e t,e Cinner glass t+beD ,aving travelle* t,ro+g, t,e entire me*i+m insi*e t,is inner t+be. 6.@.7 $f negative on t,e first *a 1 =eep t,e inoc+late* t+be at t,e room temperat+re for a f+rt,er 4 to E *a s to see if evi*ence of motilit is present. Prepare% *y Microbiologist Approve% *y Management Representative
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DOC. No. : AV/MB/SOP/002 Page No. : Page 4 of 4 00

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/.10 0est for 7rease 6.10.1 $noc+late t,e organism from 24-,o+r inc+bate* A+trient brot, c+lt+re ,eavil over t,e entire slope s+rface of me*i+m an* inc+bate at 78B" for 1? to 24 ,o+rs. 6.10.2 A positive (rease is s,o0n b t,e me*i+m becoming pin= or re* on inc+bation. $f negative1 contin+e inc+bation for at least 4 *a s. 6.10.7 Prote+s species give a positive res+lt1 an* can be +se* as a control. /.11 0est for 8,! pro%uction 6.11.1 $noc+late )S$ me*i+m b stabbing t,e strain into t,e b+tt an* strea=ing t,e slope. 6.11.2 $nc+bate at 78B" an* observe *ail for +p to 8 *a s. 6.11.7 ),e presence or absence of blac=ening in t,e b+tt of t,e me*i+m is to be recor*e*. /.1, 0est for Citrate utili+ation 6.12.1 $noc+late t,e strain on to a SimmonDs citrate agar me*i+m 0it, a o+ng A+trient agar slant c+lt+re +sing a straig,t 0ire. 6.12.2 $nc+bate at 789" for +p to 4 *a s for gro0t, of t,e organisms. /.1- 0est for (n%ole 6.17.1 $noc+late me*i+m :20g peptone an* 6g so*i+m c,lori*e in 1000 ml 0ater; 0it, a loopf+l of 24 ,o+rs gro0t, in A+trient brot, an* inc+bate at 789 " for 4? ,o+rs . 6.17.2 A** 0.6 ml =ovacDs reagent. 6.17.7 After a**ing FovacDs reagent1 s,a=e t,e t+be gentl 1 t,e appearance of a re* colo+r in*icates t,e presence of $n*ole. 2.0 Documentation: Microbiological )est Report eferences: $S 6??8 :PAR) 8;'1@@@

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