AOAC SPIFAN Stakeholder Panel Meeting Book (March 14, 2019)

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ISO 4833-1:2013 and GB 4789.2-2016

InternationalOfficialMethod (OM)

InternationalOfficialMethod

Comment leading to result difference

US FDABacteriologicalAnalyticalManual (BAM) Chapter3: AerobicPlateCount (andStandardMethods for theExaminationofDairy Products [SMEDP], 17th Ed,Chapter6)

AOACOMA986.33 (milk); 989.10 (dairy); 990.12 (foods) NFValdiationviaAFNOR Certification: 3M01/1-09/89 (3M Petrifilm AerobicCountPlate)

Methodnumber

ISO4833-1:2013

GB4789.2-2016

Analyte

aerobicbacteria

aerobicmicrobial colonies

aerobicbacteria

Products intended for human consumptionand for animal feed;environmental samples in theareaof foodand feed productionandhandling. Food Colony countat30 q C by thepourplate technique. A specifiedquantityof the liquid testsample, and/oran initial suspension in thecaseofotherproducts, is dispensed intoanemptyPetri dish andmixedwith anon-selectivemolten agarmedium. Theplatesare incubatedunder aerobic conditionsat30 q C for 72 h.Thenumberofmicroorganisms pergramorper millilitre of the testsample is calculated from thenumberof coloniesobtained in theplates containing fewer than300 colonies.

Products intended for human consumptionandenvironmental samples in theareaof foodproductionandhandling. Colony countat32 q C (liquidmilk), 35 q C (otherdairy) by thepourplate technique. A specifiedquantityof the liquid testsample, and/oran initial suspension in the caseofotherproducts, isdispensed intoanemptyPetri dish and mixedwith anon-selectivemoltenagarmedium. Theplatesare incubatedunder aerobic conditionsat32 q C (liquidmilk), 35 q C (otherdairy) for 48 h.Thenumberofmicroorganisms pergram or permL of the test sample is calculated from thenumber of colonies REWDLQHGLQWKHSODWHVFRQWDLQLQJ FRORQLHV

Products intended for human consumptionandanimal feed; environmental samples in theareaof foodand feedproductionand handling. Bydry-film technique: AOACOMAs: Colony countat32 q C (liquidmilk anddairy), 35 q C (other foods). AFNOR Certification:Colony countat 30 q C (dairy), 37 q C (foods, feedand environmental samples). 1ml of liquid testsample, and/oran initial suspension in thecaseofother products, isdispensedontoaPetrifilmplateand the inoculum spread toa 20cm2 area. AOACOMAs:Plates are incubated48 hunder aerobic conditionsat32 q C (dairy), and35 q C (foods) AFNOR Certification:Plates are incubatedat30 q C for 72 h (dairy) andat 37 q C for 48 h (foods, feed,enviro). Thenumberofmicroorganisms pergramorperml of the testsample is calculated from thenumberof coloniesobtainedon theplates containing $2$&RU $)125FRORQLHV

Comment of other International Official Methods

Scope

Colony countat36 q C by thepourplate technique.(foraquaticproduct,colony countat30 q C by the pourplate technique) A specifiedquantityof the liquid testsample, and/oran initial suspension in thecaseofother products, isdispensed intoanemptyPetri dish andmixedwith anon-selectivemolten agarmedium. Theplatesare incubatedunder aerobic conditionsat36 q C for 48 h(for aquaticproduct, 30 q C for 72 h).Thenumberofmicroorganisms pergramorpermillilitre of the testsample is calculated from the numberof coloniesobtained in theplatescontaining from 30 to300 colonies.

OMmethodusing aerobic conditionsat30 q C for 72 h. GBmethodusingaerobic conditionsat36 q C for 48 h(exceptaquaticproduct at30 q C for 72 h)

MethodPrinciple

dependsupon theproductand local legislation

Regulatoryminimum andmaximum limits

- - -

Plate countagar (PCA)

Analysis Technology

N/A N/A N/A N/A

N/A N/A Ԩ r 1 N/A

agar -pourplatemethod

dry-filmmedia (pourplatemethod replacement)

Comment leading to result difference

25-250 colonies (AOAC) 30-300 colonies (ISO)

Calibration curve concentration range

25-250 colonies

Ԩ ,30

Ԩ r 1

N/A N/A

EquipmentParameters Calibration Technique

Incubator,36

Sampleweight

1mL of liquid sample or1mL of initial suspensionofotherproducts

25mL of liquid sampleor 25 gofotherproducts

Sampleweight isdifferent

1mL of liquid sample or1mL of initial suspensionofproducts Solid test samples:weigh a representativemass (minimum 10gor as stated)of the testsample.Addaquantityofdiluentequal to9x themass andhomogenize themixture.Prepare decimaldilutionsas appropriate. Liquid testsamples: Prepare decimaldilutions asappropriate.

According to ISO6887 - solid test samples:weigh a representativemass (minimum 10gor as stated)of the testsample.Addaquantityof diluentequal to9x themass andhomogenize themixture.Prepare decimaldilutions asappropriated. Liquid testsamples: Prepare decimaldilutions asappropriated.

solid test samples:weigh a representativemass (25 g)of the testsample.Add225mL diluentequal andhomogenize themixture.Prepare decimaldilutionsas appropriated. Liquid test samples:weigh a representativemass (25mL) of the test sample.Add225mL diluent equalandhomogenize themixture.Prepare decimaldilutions asappropriated.

Sample preparation

Plate countagar (PCA)

Culturemedia

Plate countagar (PCA)

Plate countmediaw/guar

OMmethodusing aerobic conditionsat30 q C for 72 h. GBmethodusingaerobic conditionsat36 q C for 48 h(exceptaquaticproduct at30 q C for 72 h)

Transfer 1mL of test sample if liquid,or 1mL of the initial suspension î GLOXWLRQRIRWKHUSURGXFWVRQWR HPSW\3HWULILOPSODWHV,I necessary, repeat theprocedurewith furtherdecimaldilutions. Spread the inoculum toa20cm2 area,using theappropriate spreader. Incubateplatesat32 r 1 q C (dairy) or35 r 1 q C (other foods), for 48 r 3 h. (AOACOMA) Incubateplatesat30 r 1 q C for 72 r 3;andat37 r 1 q C (foods, feedand environmental samples).

Transfer 1mL of test sample if liquid,or 1mL of the initial suspension î GLOXWLRQRIRWKHUSURGXFWVLQWR HPSW\SODWHV,IQHFHVVDU\UHSHDW theprocedurewith further decimaldilutions. Pour about12mL to15mL of theplatecountagar intoeachPetri dish. Mix the inoculumwith themedium by rotating thePetri dishes.After complete solidification, invert theplatesand incubateat32 r 1 q C (dairy) or 35 r 1 q C (other foods) for 48 r 3h. Incasewheremicroorganisms whose coloniesovergrow thesurface of themedium are suspected,pour about4mL ofoverlaymediumor PCA on to the surfaceof the inoculatedmedium andallow to solidify before incubation.

7UDQVIHU P/RIWHVWVDPSOHLIOLTXLGRU P/RIWKHLQLWLDOVXVSHQVLRQ î GLOXWLRQRIRWKHU products into2 emptyplates.Select2~3 sampleswithproperdilution, repeat theprocedure. Pour about15mL to20mL of theplatecountagar intoeachPetri dish. Mix the inoculumwith themedium by rotating thePetri dishes.After completesolidification, invert theplatesand incubateat36 r 1 q C for 48 r 2h.For aquaticproduct,incubateat30 r 1 q C for 72 r 3h Incasewheremicroorganisms whose coloniesovergrow thesurface of themedium are suspected, pour about4mL ofoverlaymediumor PCA on to thesurface of the inoculatedmedium andallow to solidify before incubation.

7UDQVIHU P/RIWHVWVDPSOHLIOLTXLGRU P/RIWKHLQLWLDOVXVSHQVLRQ î GLOXWLRQRIRWKHUSURGXFWVLQWR HPSW\ plates. Ifnecessary, repeat theprocedurewith furtherdecimaldilutions. Pour about12mL to15mL of theplatecountagar intoeachPetri dish. Mix the inoculumwith themedium by rotating thePetri dishes.After completesolidification, invert theplatesand incubateat30 r 1 q C for 72 r 3h. Incasewheremicroorganisms whose coloniesovergrow thesurfaceof the medium are suspected,pour about4mL ofoverlaymediumor PCAon to thesurfaceof the inoculatedmedium and allow to solidify before incubation.

Procedure

Possible differences between OM/GB results (i.e due to sample weight, Incubation temperature and Incubation time). Results may be overestimated by OM.

Confirmatory steps (micro)

N/A

Countall colonies, regardless of sizeor shape

Weighing sampling is reported inCFU/g andvolume sampling is reported inCFU/mL. Countall colonies, regardless of sizeor shape

Countall colonies, regardless of sizeor shape

Countall red colonies regardless of sizeor intensity.

Performance characteristics (micro)

Report all countscomputed from duplicateplates containingcounts from 25-250 colonies/plate. Report counts less than25 ormore than250 coloniesperplateas estimatedaerobicplatecounts (EAPC).

Plateswithmore then30 and fewer then300 coloniesperplate. Calculate thenumberNofmicroorganisms present in the testsample as aweightedmean from two

Plateswithmore then10 and fewer then300 coloniesperplate. Calculate thenumberNofmicroorganisms present in the testsample as aweightedmean from twoplatesor two successivedilutions. Report as xUFC/mL or xUFC/g

Final reportable results

platesor two successivedilutions. Report as xCFU/mL or xCFU/g 1 &Q Q G

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LOD& LOQ

N/A

From SMEDPChapter1,Table1.1,basedonOMAstudydata: Relative StandardDeviation [r] (%): Rawmilk: 3.0 r 2.1 Raw&Homog.Milk= 1.5 r 0.5 Homogmilk=5.15 r 1.4 Milks=1.2 r 0.6 Chocolatemilk= 5.7 r 3.1 Cream=2.4 r 1.1 Evaporated=10.5 r 9.7 NonfatDryMilk=3.0 r 0.4 Cheese=4.2 r 1.2 Relative StandardDeviation [R] (%): Rawmilk: 9.7 r 4.2 Raw&Homog.Milk= 2.1 r 0.3 Homogmilk=12.1 r 0.9 Milks=1.5 r 0.6 Chocolatemilk= 7.4 r 3.3 Cream=9.3 r 3.4 Evaporated=10.6 r 9.6 NonfatDryMilk=5.0 r 1.4 Cheese=5.9 r 0.7

Repeatability limit, r=0,25, in log10N,whereN is the numberofmicroorganisms permillilitre Reproducibility limit,R= 0,45, in log10N,whereN is thenumberofmicroorganisms permillilitre

From SMEDPChapter1,Table1.1,basedonOMAstudydata: Relative StandardDeviation [r] (%): Raw&Homog.Milk= 1.2 r 0.4

Chocolatemilk= 4.3 r 1.7 Evaporatedmilk=8.9 r 7.3 IceCream= 6.2 r 5.9 NonfatDryMilk=4.60 r 1.2 Cheese=3.1 r 1.2 Relative StandardDeviation [R] (%): Raw&Homog.Milk= 1.8 r 0.5 Chocolatemilk= 6.8 r 2.0 Evaporatedmilk=9.2 r 6.9 IceCream= 8.2 r 5.7 NonfatDryMilk=7.0 r 2.0 Cheese=3.6 r 0.8

N/A

Precison (Repeatbility,Reproducibility)

Forpurchase (copyrighted): https://www.iso.org/standard/53728.html

CFSA online: http://bz.cfsa.net.cn/staticPages/F23DE741-721F-4C68-BDB1-F42440C66858.html; http://www.doc88.com/p-9307839386714.html

FDABAMonline: https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/uc m063346.htm

JournalAOAC INTERNATIONAL: 989.10Dairy: https://multimedia.3m.com/mws/media/393522O/dry- rehydratable-film-total-aerobic-bacteria-in-dairy-aoac-cs.pdf; 986.33Milk: https://multimedia.3m.com/mws/media/393521O/enumeration-total- bacteria-coliforms-in-milk-aoac-cs.pdf 990.12 Foods:https://multimedia.3m.com/mws/media/393523O/dry- rehydratable-film-total-aerobic-bacteria-in-foods-aoac-cs.pdf AFNOR 3M01/1-09/89 (all foods): https://nf-validation.afnor.org//en/wp- content/uploads/sites/2/2014/03/Synt-3M-01-01-09-89_en.pdf

Conclusion:Possible differences betweenOM/GB results (i.edue to sampleweight, Incubation temperature and Incubationtime).Resultsmay beoverestimatedbyOM. AlthoughGBand ISO/BAM/AOACmethods forplatecountarequitesimilar, andprobably deliver thesame answer, it ispreferredtohaveeither ISO/AOAC/BAMbecause theyhaveproven their fitness forpurposewithWELL PUBLISHED validationdata, following ISO andAOAC standards, collaborative study and review by independent,vetted,ExpertReview Panels/TechnicalCommittees andbecauseof this recognizedmethodsglobally

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Continue the sub-tasks including the evaluation and validation of Type B and C Continue the methods comparison between GB and International standards, in infant formula and dairy products (minerals, vitamins, microbiological etc) Organize the high-level meeting at AOAC China meeting to facilitate the collaboration between GB and AOAC/ISO/IDF Organize the workshop at AOAC China meeting to further discuss the comparison of Vitamin D and Aerobic Plate Count and align on the next step CSIQ set up China association standard Committee (on food) to formulate association standard, include adopt international standard. International experts are invited to contribute to this platform.

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