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1 3M Petrifilm Salmonella Express System MJ Shin, 3M Korea

2 Petrifilm Salmonella Express System Petrifilm salmonella Express System 이란? 정성분석시스템 ( 증균 필름 디스크 ) 신속검출, 생물화학적검증가능 증균배지, 증균 supplement, 필름배지, 확정디스크로이루어짐 증균후필름으로추정결과가능 확정필요시디스크사용 제공된배지사용하여야함 식품샘플외화장품, 제약, 수의학, 인체감염검증등에사용할수없음. 2 Confidential Do not copy or distribute outside of 3M

3 검사방법요약 2 가지증균배양법 : High microbial background vs Low microbial background 2 가지증균배양법모두 3M Salmonella Enrichment Broth + 3M Salmonella Enrichment Supplement 사용 High microbial background 인경우만 R-V R10 사용 Single streak method Single incubation temperature of 41.5 ± 1 C 3

4 4 3M Food Safety

5 외부기관실험결과 ( 미 Q lab) 실험한식품군에대하여는비교방법과동일한결과 위양성없었음 한번스트리킹하는방법이기존스트리킹방법 (4 번 ) 보다수월 결과분석및판독이수월 비교방법대비빠른결과 5

6 Competitive Analysis 3M Petrifilm SALX Plate Petrifilm SALX Plate Powdered or Pre-poured Agar (Selective/ Differential) Powdered or Prepoured Chromogenic agar Petrifilm Me-Too Plate appearance Company Time to Presumptive Result Time to Confirmed Result from Presumptive Validation Level of detection 3M Food Safety Nissui, Oxoid Merck, BD, AES, BioMerieux Oxoid, BD, BioMerieux Chisso Sanita-kun Nissui Compact Dry hrs hrs 37-58hrs hrs 4 hrs hrs hrs hrs AOAC PTM, In progress: AOAC OMA, MicroVal Biochemical Confirmation Reference Method Presumptive Positive; agar is used with one other media per reference method. BD = AOAC; BioMerieux IBISA = AFNOR Presumptive Positive None Presumptive Positive 6

7 7 3M Food Safety

8 8 3M Food Safety

9 9 3M Food Safety

10 3M Petrifilm Salmonella Express System Shelf Life & Storage Temperature 10

11 3M Petrifilm Salmonella Express System Estimated Number of Tests Per Packout Catalog Number 70# Product Name Packout TOTAL TESTS (Based on 225 ml) M Petrifilm Salmonella Express Plate 25 plates/ pouch, 2 pouches /box M Petrifilm Salmonella Express Plate 25 plates/ pouch, 8 pouches /case M Petrifilm Salmonella Express Confirmation Disk 5 disks/ pouch, 1 pouches /box M Petrifilm Salmonella Express Confirmation Disk 5 disks/ pouch, 5 pouches /case 25 SEB M Salmonella Enrichment Base 500g/bottle 1 ea 60 SEB M Salmonella Enrichment Base 2.5kg/bottle 1ea 300 SESUP M Salmonella Enrichment Supplement 1g/vial 1ea 88 BPO M R-V R10 (Secondary Enrichment for High Microbial Load samples only) 500g/bottle

12 Indicator Petrifilm vs Pathogen Petrifilm Petrifilm Indicator Quantitative Plates Petrifilm Pathogen Qualitative SALX Plate Type of Test Indicator quantitative, looking for colony counts Pathogen qualitative, looking for presence/ absence Plate Hydration Only for environmental air and surface sampling and membrane filtration technique (Petrifilm Aqua) Must hydrate plate prior to use Enrichment N/A 3M Base, Supp, (R-V R10 for high background food only) required Plate Inoculation Use pipette and dispense onto plate Streak sample with loop onto hydrated gel plate Shelf Life 18 months 10 months at launch 18 months (8 months post launch) Shipping Ambient Refrigerated (2-8 deg. C) required Storage Confirmation Method Value Prop Store unopened pouches in refrigerator upon receipt. Open pouches at room temperature for maximum 30 days. Coliforms and E.coli confirmed via gas bubble formation on plate. STX plate and disk process validated vs. BAM method (including confirmation step) Ready made media Greater Consistency Longer shelf life than pre-made agar dishes Takes up less space than agar dishes Store unopened pouches Refrigerated (2-8 deg. C) upon receipt To prevent exposure to moisture, store opened 3M Petrifilm SALX Plate pouches in a sealed bag, protected from light, at -20 to - 10 C for no longer than 4 weeks. Biochemical Confirmation method with disk Must circle presumptive positive colonies prior to adding Confirmation Disk Ready made media Greater Consistency Longer shelf life than pre-made agar dishes Takes up less space than agar dishes Fast time to presumptive & confirmation results All in one test method presumptive & Biochemical Confirmation 12

13 3M Method : Enrichment media 3M Salmonella Enrichment Broth with 3M Salmonella Enrichment Supplement 1 차증균 = hours High background samples 2 차증균 = R-V R10; 0.1 ml into 10 ml incubate for 8-24 hrs. Temp. = 41.5 C ± 1 Key Attribute 3M SALX System method (combination of SEB +Supplement +Plate + Disk = System) 를을모두사용시살모넬라의생장을높이고다른미생물의생장은저해함 13 3M All Rights Reserved.

14 3M Salmonella Petrifilm System 검사방법 / Key 특징 3M Salmonella Enrichment Base Key 특징 Sterilized via steam autoclave (121 C for 15 minutes) ph 보정필요하지않음 멸균후어두운황색으로변함 일반적미생물실험실에서사용되는기기및기구사용 만약멸균후 ph 가맞지않는다면사용불가 과멸균이되었을가능성있음. 선택성과민감도가떨어지게됨 DO NOT ph adjust 14 3M All Rights Reserved.

16 Media 제조법 : 37.0 g 1.0 L. Autoclave 121 C,15 Supplement( ):. 50 mg ( 2.5 mg) (20 to 42.5 C ). ph 7.0 ± 0.2 at 25 C. 16

17 17 3M Food Safety Media 유효기간

18 3M Salmonella Petrifilm System 검사방법 / Key 특징 Secondary Enrichment: R-V R10 Can use any R-V R10 as long as formulation is same as specified in IFU Only required for HIGH Microbial Background Samples Incubate for 8-24 hrs. Temp. = 41.5 C ± 1 Key Attribute R-V R10 formula = RV 보다약간낮은 ph 살모넬라선택증균 8 ~ 24 시간증균의유의적차이없음 실험스케줄에맞추어조정가능 18 3M All Rights Reserved.

19 3M Salmonella Petrifilm System 검사방법 / Key 특징 Plate hydration Key Attribute 2 ml sterile RO water, distilled water, Butterfield s Phosphate Diluent 젤화시간 : 상온 1시간 Use 3M Flat Spreader 냉동보관가능 1 hour gel time = same as 3M Petrifilm AQUA 3M Petrifilm Staph Express Spreader로혼용가능 젤화된필름은상온에서 8 시간방치가능 -20 ~ -10 C 5 일까지보관가능 Hydrated Gel Color Check = 컬러카드로바로확인가능 19 3M All Rights Reserved.

20 Hydrated Gel Plate Color Card A Hydrated Gel Plate Color Card 는필름박스에포함 운송중고온이노출되었을가능성을확인하기위해필름젤화후색변화확인 희석액으로젤화후적색이나밝은분홍색이면정상 희석액으로젤화후주황색이나갈색이면사용불가 20

22 3M Salmonella Petrifilm System 검사방법 / Key 특징 Plate Inoculation 10 ul loop (sterile plastic, new metal, or smooth metal loop) Single streak for isolation Incubation time = 24 hrs ± 2 hrs. Incubation temp. = 41.5 C ± 1 Key Attribute 10 ul loop size 로접종하였을때민감도및결과가유효 적게접종되었을때는민감도보장어려움 Single streak : quadrant streak, 보다편리 22 3M All Rights Reserved.

23 3M Salmonella Petrifilm System 검사방법 / Key 특징 Presumptive Positives 푸른콜로니, 녹색콜로니, 청록색콜로니, 검정콜로니 ( 황색존이나가스방을이있는경우에도추정콜로니아님 ) 적색, 적갈색, 진적색콜로니여도황색존이나가스방울없으면추정콜로니아님 적갯, 적갈색, 진적색콜로니에적색존이있는경우도추정콜로니아님 확정디스크삽입전에꼭! 추정콜로니에표시를해야함 적어도 5개이상의콜로니를표시 Key Attribute 콜로니색컴정표를이용하여검정가능 Petrifilm EB Plate: yellow acid zone / gas bubble 와비슷한해석방법 콜로니카운터사용시가스방울은구별하기쉬우나황색존은구별어려움. 23 3M All Rights Reserved.

24 판독카드 Side 1 24 Side 2

25 3M Salmonella Petrifilm System 검사방법 / Key 특징 Biochemical Confirmation 추정콜로니에표시한후확정디스크삽입 Incubate at 41.5 C ± 1 C for 4-5 hours 표시된몰로니의색변화만을관찰하여판단 녹색, 청록색, 파란침전물이있는청색, 감청색, 검정색 Key Attribute 디스크는앞뒤구분없음 표시한콜로니만확인하면되므로추가분석간소화 25 3M All Rights Reserved.

26 Method Approvals AOAC PTM and OMA ISO AOAC PTM = estimated 1Q 2013 AOAC OMA = estimated 4Q 2013 Microval = estimated 4Q M is pursuing method approvals recognized globally 26

27 A Comparative Evaluation of the 3M Petrifilm Salmonella Express System for the Detection of Salmonella species in Food and Environmental Surfaces AOAC Method Validation 외부공인기관에서실험 아래의기존의기준실험방법과비교실험 USDA/FSIS-MLG 4.05 (2011) Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg and Catfish Products FDA/BAM Chapter 5 Salmonella method ISO 6579: Microbiology of food and animal feeding stuffs-horizontal method for the detection of Salmonella spp. 27

28 Method Validation Procedure Summary: 식품당 20 번반복 One inoculum level: CFU/test portion Fractional Food Testing 5 개의음성대조군 : 0 CFU/test portion (un-inoculated) 통계분석 : Mantel-Haenszel chi square analysis for unpaired test portions and by Probability of Detection (POD) 28 3M All Rights Reserved.

29 Organisms for Inoculation: Matrix Inoculating Organism Matrix Inoculating Organism Raw Ground Chicken Salmonella enterica spp. Heidelberg NCTC 5717 Frozen, Uncooked Shrimp Salmonella enterica spp. Virchow ATCC Pasteurized Liquid Whole Eggs Salmonella enterica spp. Enteritidis ATCC Fresh Spinach Salmonella enterica spp. Saintpaul ATCC 9712 Dry Dog Food Salmonella enterica spp. Poona NCTC 4840 Raw Ground Beef Salmonella enterica spp. Ohio STS 81* Raw Ground Pork Salmonella enterica spp. Montevideo ATCC 8387 Cooked Chicken Nuggets Salmonella enterica spp. Typhimurium ATCC Stainless Steel Environmental Surface Salmonella enterica spp. Kahla ATCC M All Rights Reserved.

30 Fractional Food Matrices & Comparative Results Food matrix Raw ground chicken PetrifilmSalmonella Express Presumptive/Confirmed Reference Method (BAM or USDA) Presumptive /Confirmed ISO 6579 Reference method 15/15 14/14 14/14 Liquid whole egg 6/6 4/4 5/5 Fresh frozen shrimp 12/12 10/10 Spinach 11/11 10/10 Dry dog food 16/16 14/14 Raw ground beef 13/13 13/13 Frozen chicken nuggets 6/6 6/6 Raw ground pork 9/9 9/9 Stainless Steel 7/7 6/6 30

31 Competitive Analysis 3M SALX System 3M Food Safety Petrifilm SALX Plate Plate appearance Powdered or Pre-poured Agar (Selective/ Differential) Powdered or Prepoured Chromogenic agar 31 Company Time to Presumptive Result Time to Confirmed Result from Presumptive Validation Method Limitations Level of detection 3M Food Safety Nissui, Oxoid Merck, BD, AES, BioMerieux Oxoid, BD, BioMerieux hrs hrs hrs 4 hrs hrs hrs AOAC PTM, AOAC OMA, MicroVal Aytpical lactose positive strains (S. arizonae and S. diarizonae) Biochemical Confirmation Reference Method Brilliant Green agar = S. typhi and S. paratyphi Presumptive Positive; agar is used with one other media per reference method. BD = AOAC; BioMerieux IBISA = AFNOR S. typhi, S. paratyphi A, S. typhimurium, S. choleraesuis, S. minnesota, S. enterica subsp. arizonae, S. pullorum. S. Dublin may not grow for some media Presumptive Positive

32 공 Salmonella 25 g or 25 ml MacConkey DC XLD 24 2h 2h / 35~37 BS 225 ml h / 35~37 Ratio 1:100 Rapparport(1956) 0.1 ml 10 ml RV NA 18~24 h / 35~37 TSI 18~24 h / 35~37 Vassiliadis(1976) h 2h / 42 1 Gram stain (-) Urease (-) Lys decarboxylase (+) Etc. TECRA for Auto/manual identification 32 32

33 Biochemical Confirmation 디스크를이용한생화학적확정방법은타생화학적방법과동일한결과를갖음. 비교방법 TSI, 그램염색등일반생화학법시험법 Vitek or API O&H 항형철형시험법과비교하지않음 33

34 고려사항 Some atypical, 유당분해양성살모넬라종은다른유당분해양성미생물과구별되지않을수있음 (less than 1% of the total Salmonella serotypes). (S. arizonae and S. diarizonae) 34

35 35 3M All Rights 18~ ±1. 누 9 다른미생물오염이심할시 0.1ml 배양액을 10ml 의 RVR10 배지로옮긴다 Confidential Do not copy or distribute outside of 3M RVR10 배지를 18~ 24 시간동안 41.5±1 도에서배양.

36 36 3M All Rights 2ml 8 20~25. 누, 누 3, 누 Butterfield s Phosphate Diluent 등. Confidential Do not copy or distribute outside of 3M 20~25

37 10ul 누 loop. 히 ) 10ul 누 loop. 히 ). 누히 ) 37 3M All Rights Confidential Do not copy or distribute outside of 3M

38 . 41.5±1 24±2. 누 20 ). 누 히 ) 38 3M All Rights

39 . 누. 누. 41.5±1 4~5. 누. 누. 39 3M All Rights Confidential Do not copy or distribute outside of 3M

40 살모넬라콜로니 적색콜로니에황색존, 가스방울혹은모두존재 디스크반응후진한청색콜로니, 혹은붉은가운데진청색침전물 40 Confidential Do not copy or distribute outside of 3M

41 Identifying Salmonella Species The following are presumptive positive Salmonella species: Non- Salmonella species,,, ( ),,,, 41 Must circle presumptive positive Salmonella colonies prior to disking

42 다른미생물콜로니 황색존없는붉은콜로니, 붉은갈색존의붉은콜로니 가스방울없음 디스크반응후붉은콜로니색그대로이고진청색침전물이없음 1. 붉은콜로니, 황색존, 버블없음 2. 청록색콜로니에가스버블없음 42 3M All Rights Confidential Do not copy or distribute outside of 3M

43 Plate Sample 43

44 44 3M All Rights 3M Food Safety

45 45 3M Food Safety

46 46 3M All Rights 3M Food Safety

47 47 3M Food Safety

48 48 3M All Rights 3M Food Safety

49 49 3M Food Safety

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( )Kju225.hwp 비임균성비뇨생식기감염에서 의진단적효용성 Usefulness of the Mycofast Test (MYCOFAST Evolution 2) for the Diagnosis of Nongonococcal Genitourinary Infections Hang Ro Park, Yang Hyun Kim, Ho Jae Lee, Jea Sang Oh, Hyoung Jin