3m food safety 3m petrifilm salmonella express...

3M Food Safety

3M™ Petrifilm™ Salmonella Express System3M Food Safety

3M™ Petrifilm™ Salmonella Express System

MJ Shin, 3M Korea

3M Food Safety3M Food Safety PetrifilmPetrifilm SalmonellaSalmonella Express SystemExpress System

PetrifilmPetrifilm salmonellasalmonella Express System Express System 이란이란이란이란이란이란이란이란??

�정성분석시스템 (증균�필름�디스크)

�신속검출, 생물화학적검증가능

�증균배지, 증균 supplement, 필름배지, 확정디스크로이루어짐

�증균후필름으로추정결과가능

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Confidential – Do not copy or distribute outside of 3M

�증균후필름으로추정결과가능

�확정필요시디스크사용

�제공된배지사용하여야함

�식품샘플외화장품, 제약, 수의학, 인체감염검증등에사용할수없음.

3M Food Safety3M Food Safety

� 2 가지증균배양법 :

� High microbial background vs

� Low microbial background

� 2가지가지가지가지증균증균증균증균배양법배양법배양법배양법모두모두모두모두� 3M Salmonella Enrichment Broth

� + 3M Salmonella Enrichment Supplement 사용

검사방법요약

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� + 3M Salmonella Enrichment Supplement 사용

� High microbial background 인인인인경우만경우만경우만경우만 R-V R10 사용

� Single streak method

� Single incubation temperature of 41.5 ± 1°C


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외부외부외부외부기관기관기관기관실험실험실험실험결과결과결과결과 (미미미미 Q lab)

•실험한식품군에대하여는비교방법과동일한결과

•위양성없었음

•한번스트리킹하는방법이기존스트리킹방법(4번) 보다 수월

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•결과분석및판독이수월

•비교방법대비빠른결과


Competitive Analysis – 3M Petrifilm SALX Plate

Petrifilm SALX Plate Powdered or Pre-poured

Agar (Selective/

Differential)

Powdered or Pre-

poured

Chromogenic agar

Petrifilm

“Me-Too”

Plate

appearance

Company

3M Food Safety

Nissui, Oxoid

Merck, BD, AES,

BioMerieux

Oxoid, BD, BioMerieuxChisso “Sanita-kun”

Nissui ”Compact Dry”

Time to

6

Time to

Presumptive

Result

40-48 hrs 72-96 hrs 37 - 58hrs 24 - 60 hrs

Time to

Confirmed

Result from

Presumptive

4 hrs 54 – 78 hrs 59 - 102 hrs 24-48 hrs

Validation AOAC – PTM,

In progress: AOAC OMA,

MicroVal

Reference Method

BD = AOAC;

BioMerieux IBISA =

AFNOR

None

Level of

detection Biochemical ConfirmationPresumptive Positive; agar is

used with one other media per

reference method.

Presumptive Positive Presumptive Positive


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8


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Shelf Life & Storage Temperature

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Catalog

Number 70# Product Name Packout

TOTAL TESTS

(Based on

225 mL)

6536 70-2007-6998-5 3M Petrifilm Salmonella Express Plate 25 plates/ pouch, 2 pouches /box 50

6537 70-2007-6999-3 3M Petrifilm Salmonella Express Plate 25 plates/ pouch, 8 pouches /case 200

6538 70-2007-7000-9 3M Petrifilm Salmonella Express Confirmation Disk 5 disks/ pouch, 1 pouches /box 5


Estimated Number of Tests Per Packout

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6538 70-2007-7000-9 3M Petrifilm Salmonella Express Confirmation Disk 5 disks/ pouch, 1 pouches /box 5

6539 70-2007-7001-7 3M Petrifilm Salmonella Express Confirmation Disk 5 disks/ pouch, 5 pouches /case 25

SEB500 70-2007-7477-9 3M Salmonella Enrichment Base 500g/bottle 1 ea 60

SEB025 70-2007-7478-7 3M Salmonella Enrichment Base 2.5kg/bottle 1ea 300

SESUP001 70-2007-7435-7 3M Salmonella Enrichment Supplement 1g/vial 1ea 88

BPO288500 70-2007-5512-5 3M R-V R10

(Secondary Enrichment for High Microbial Load samples only)500g/bottle 1880


Indicator Petrifilm vs Pathogen PetrifilmPetrifilm 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

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Shipping Ambient Refrigerated (2-8 deg. C) required

Storage • Store unopened pouches in refrigerator upon receipt.

• Open pouches at room temperature for maximum 30 days.

• 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.

Confirmation

Method

• Coliforms and E.coli confirmed via gas bubble formation on

plate.

• STX plate and disk process validated vs. BAM method

(including confirmation step)

• Biochemical Confirmation method with disk

• Must circle presumptive positive colonies prior to adding

Confirmation Disk

Value Prop • Ready made media

• Greater Consistency

• Longer shelf life than pre-made agar dishes

• Takes up less space than agar dishes

• 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


Enrichment media

• 3M Salmonella Enrichment Broth with 3M SalmonellaEnrichment Supplement

• 1 차증균= 18-24

Key Attribute

• 3M SALX System method (combination of SEB +Supplement +Plate + Disk = System) 를을모두사용시살모넬라의

3M Method :

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© 3M 2007. All Rights Reserved.

• 1 차증균= 18-24 hours

• High background samples 2 차증균= R-V R10; 0.1 mLinto 10 mL incubate for 8-24 hrs.

• Temp. = 41.5 °C ± 1

사용시살모넬라의생장을높이고다른미생물의생장은저해함


3M SalmonellaEnrichment Base

• Sterilized via steam autoclave (121 °C for 15 minutes)

• pH 보정필요하지않음 –

Key 특징특징특징특징

• 일반적미생물실험실에서사용되는기기및기구사용

• 만약멸균후 pH 가맞지않는다면사용불가

3M Salmonella Petrifilm System 검사방법 / Key 특징

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• pH 보정필요하지않음 –

• 멸균후어두운황색으로변함

않는다면사용불가

• 과멸균이되었을가능성있음.

선택성과민감도가떨어지게됨

• DO NOT pH adjust


High Background와 Low Background의차이

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Media 제조법

� :

37.0 g 1.0 L . Autoclave 121°°°°C ,15

�Supplement( ):

• .

• 50 mg (±±±± 2.5 mg) (20 to

42.5°C ) .

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42.5°C ) .

pH 7.0 ± 0.2 at 25°C.


Media 유효기간

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Secondary Enrichment: R-V R10

• Can use any R-V R10 as long as formulation is same as specified in IFU

Key Attribute

• R-V R10 formula = RV 보다약간낮은 pH

• 살모넬라선택증균 8


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• Only required for HIGH Microbial Background Samples

• Incubate for 8-24 hrs.

• Temp. = 41.5 °C ± 1

• 살모넬라선택증균 8

~ 24 시간증균의유의적차이없음

• 실험스케줄에맞추어조정가능


Plate hydration

• 2 mL sterile RO water, distilled water, Butterfield’s Phosphate Diluent

• 젤화시간 : 상온

Key Attribute

• 1 hour gel time = same as 3M Petrifilm AQUA

• 3M Petrifilm Staph Express Spreader로혼용가능


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• 젤화시간 : 상온1시간

• Use 3M Flat Spreader

• 냉동보관가능

혼용가능

• 젤화된필름은상온에서 8 시간방치가능

• -20 ~ -10°C 5일까지보관가능

• Hydrated Gel Color Check = 컬러카드로바로확인가능


Hydrated Gel Plate Color Card

� A Hydrated Gel Plate Color Card는필름박스에포함

� 운송중고온이노출되었을가능성을확인하기위해필름젤화후색변화확인

� 희석액으로젤화후적색이나밝은분홍색이면정상

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분홍색이면정상

� 희석액으로젤화후주황색이나갈색이면사용불가


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© 3M 2011. All Rights Reserved. Confidential – Do not copy or distribute outside of 3M


Plate Inoculation

• 10 uL loop (sterile plastic, new metal, or smooth metal loop)

• Single streak for isolation

Key Attribute

• 10 uL loop size 로접종하였을때민감도및결과가유효

• 적게


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• Single streak for isolation

• Incubation time = 24 hrs ± 2 hrs.

• Incubation temp. = 41.5 °C ± 1

• 적게접종되었을때는민감도보장어려움

• Single streak : quadrant streak, 보다편리


Presumptive Positives

• 푸른콜로니, 녹색콜로니, 청록색콜로니, 검정콜로니 (황색존이나가스방을이있는경우에도추정콜로니아님)

• 적색, 적갈색, 진적색콜로니여도황색존이나가스방울없으면

Key Attribute

• 콜로니색컴정표를이용하여검정가능

• Petrifilm EB Plate: yellow acid zone / gas bubble 와비슷한해석방법

• 콜로니카운터사용시가스방울은구별하기쉬우나황색존은구별어려움.


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가스방울없으면추정콜로니아님

• 적갯, 적갈색, 진적색콜로니에적색존이있는경우도추정콜로니아님

• 확정디스크삽입전에꼭! 추정콜로니에표시를해야함

• 적어도 5개이상의콜로니를표시

황색존은구별어려움.


판독카드

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Side 1

Side 2


Biochemical Confirmation

• 추정콜로니에표시한후확정디스크삽입

• Incubate at 41.5°C ±1°C for 4 - 5 hours

• 표시된몰로니의색

Key Attribute

• 디스크는앞뒤구분없음

• 표시한콜로니만확인하면되므로추가분석간소화


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• 표시된몰로니의색변화만을관찰하여판단

• 녹색, 청록색,

• 파란침전물이있는청색, 감청색, 검정색


Method Approvals

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AOAC PTM and OMA ISO 16140

3M is pursuing method approvals recognized globally

AOAC PTM = estimated 1Q 2013AOAC OMA = estimated 4Q 2013 Microval = estimated 4Q 2013


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

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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.


•식품당 20번반복

•One inoculum level: 0.2-2 CFU/test portion – Fractional Food

Testing

• 5개의음성대조군 : 0 CFU/test portion (un-inoculated)

Method Validation Procedure Summary:

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• 5개의음성대조군 : 0 CFU/test portion (un-inoculated)

•통계분석 : Mantel-Haenszel chi square analysis for unpaired

test portions and by Probability of Detection (POD)


Matrix Inoculating Organism Matrix Inoculating Organism

Raw Ground ChickenSalmonella enterica spp.

Heidelberg NCTC 5717

Frozen, Uncooked Shrimp

Salmonella enterica

spp. VirchowATCC 51955

Pasteurized Liquid Whole Eggs

Salmonella enterica spp. Enteritidis

ATCC 13076Fresh Spinach

Salmonella enterica

spp. SaintpaulATCC 9712

Organisms for Inoculation:

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Dry Dog FoodSalmonella enterica spp. Poona

NCTC 4840Raw Ground Beef

Salmonella enterica

spp. OhioSTS 81*

Raw Ground PorkSalmonella enterica spp.

MontevideoATCC 8387

Cooked Chicken Nuggets

Salmonella enterica

spp. TyphimuriumATCC 14028

Stainless Steel Environmental Surface

Salmonella enterica spp. KahlaATCC 17980


Fractional Food Matrices & Comparative Results

Food matrixPetrifilm Salmonella Express

Presumptive/Confirmed

Reference Method (BAM or

USDA)

Presumptive /Confirmed

ISO 6579 Reference method

Raw ground

chicken15/15 14/14 14/14

Liquid whole egg 6/6 4/4 5/5

Fresh frozen

shrimp12/12 10/10

Spinach 11/11 10/10

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Spinach 11/11 10/10

Dry dog food 16/16 14/14

Raw ground beef 13/13 13/13

Frozen chicken

nuggets6/6 6/6

Raw ground pork 9/9 9/9

Stainless Steel7/7 6/6

3M Food Safety3M Food SafetyCompetitive Analysis – 3M SALX SystemPetrifilm SALX Plate Powdered or Pre-poured Agar

(Selective/

Differential)

Powdered or Pre-

poured Chromogenic

agar

Plate

appearance

Company

3M Food Safety

Nissui, Oxoid

Merck, BD, AES,

BioMerieux

Oxoid, BD, BioMerieux

Time to

Presumptive

Result

40-48 hrs 72-96 hrs 37 - 58 hrs

Time to

Confirmed

31

Confirmed

Result from

Presumptive

4 hrs 54 – 78 hrs 59 - 102 hrs

ValidationAOAC – PTM, AOAC

OMA, MicroValReference Method

BD = AOAC;

BioMerieux IBISA =

AFNOR

Method

Limitations

Aytpical lactose positive

strains (S. arizonae and

S. diarizonae)

Brilliant Green agar = S. typhi

and S. paratyphi

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

Level of

detection Biochemical ConfirmationPresumptive Positive; agar is

used with one other media per

reference method.

Presumptive Positive


225 ml 225 ml 225 ml 225 ml

25 g or 25 ml25 g or 25 ml25 g or 25 ml25 g or 25 ml MacConkeyMacConkeyMacConkeyMacConkey XLDXLDXLDXLDDCDCDCDC

24242424 2h / 35~372h / 35~372h / 35~372h / 35~37

BSBSBSBS

Salmonella

공공공공공공공공

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24242424 2 h / 35~372 h / 35~372 h / 35~372 h / 35~37

0.1 ml0.1 ml0.1 ml0.1 ml

10 ml 10 ml 10 ml 10 ml RV RV RV RV

24242424 2h / 2h / 2h / 2h / 42424242 1111

TSI TSI TSI TSI

18~24 h / 18~24 h / 18~24 h / 18~24 h / 35~3735~3735~3735~37

Ratio 1:100Rapparport(1956)

℃ ℃

Vassiliadis(1976)43℃ → 42℃

• Gram stain (-)• Urease (-)• Lys decarboxylase (+)• Etc.

NANANANA

18~24 h / 18~24 h / 18~24 h / 18~24 h / 35~3735~3735~3735~37

TECRA for

Auto/manual

identification


Biochemical Confirmation

� 디스크를이용한생화학적확정방법은타생화학적방법과동일한결과를갖음.

� 비교비교비교비교방법방법방법방법

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� 비교비교비교비교방법방법방법방법

TSI, 그램염색등일반생화학법시험법

Vitek or API

O&H 항형철형시험법과비교하지않음


고려사항

� Some atypical, 유당유당유당유당분해분해분해분해양성양성양성양성살모넬라살모넬라살모넬라살모넬라종은종은종은종은다른다른다른다른유당유당유당유당분해분해분해분해양성양성양성양성미생물과미생물과미생물과미생물과구별되지구별되지구별되지구별되지않을않을않을않을수수수수있음있음있음있음 (less than 1% of the total Salmonellaserotypes). (S. arizonae and S. diarizonae)

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살모넬라콜로니

적색콜로니에황색존,

가스방울혹은모두존재

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Confidential – Do not copy or distribute outside of 3M

디스크반응후진한청색콜로니,

혹은붉은가운데진청색침전물


Identifying Salmonella SpeciesThe following are presumptive positive Salmonella species:

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Non- Salmonella species

• , , ,

(

)

• , ,

• , ,

Must circle presumptive positive Salmonella colonies prior to disking


Plate Sample

43


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