CERTIFICATION. Certificate No. The AOAC Research Institute hereby certifies that the performance of the test kit known as:

Transcription

1 CERTIFICATION AOAC Performance Tested SM Certificate No The AOAC Research Institute hereby certifies that the performance of the test kit known as: MC Media Pad E. coli/coliform manufactured by JNC Corportation 2 1, Otemachi 2 chome Chiyoda ku Tokyo Japan This method has been evaluated in the AOAC Performance Tested Methods SM Program, and found to perform as stated by the manufacturer contingent to the comments contained in the manuscript. This certificate means that an AOAC Certification Mark License Agreement has been executed which authorizes the manufacturer to display the AOAC Performance Tested SM certification mark along with the statement "THIS METHOD'S PERFORMANCE WAS REVIEWED BY AOAC RESEARCH INSTITUTE AND WAS FOUND TO PERFORM TO THE MANUFACTURER'S SPECIFICATIONS" on the above mentioned method for a period of one calendar year from the date of this certificate (January 14, 2018 December 31, 2018). Renewal may be granted at the end of one year under the rules stated in the licensing agreement. Deborah McKenzie Deborah McKenzie, Senior Director Signature for AOAC Research Institute January 14, 2018 Date 2275 Research Blvd., Ste. 300, Rockville, Maryland, USA Telephone: Fax: Internet e mail: aoacri@aoac.org * World Wide Web Site:

2 METHOD AUTHORS Masashi Ushiyama and Mihoko Iwasaki SUBMITTING COMPANY Chisso Corporation, Otemachi Chiyoda ku, Tokyo Japan CURRENT SPONSOR JNC Corporation 2 1, Otemachi 2 chome Chiyoda ku Tokyo Japan KIT NAME(S) MC Media Pad E. coli/coliform CATALOG NUMBERS 1000 plates; code SK97A25 (25 plates x 40), SK97A10 (10 plates x 100) 100 plates; code SK97B25 (25 plates x 4), SK97B10 (10 plates x 10) INDEPENDENT LABORATORY Silliker, Inc., Food Science Center 160 Armory Drive South Holland, IL USA AOAC EXPERTS AND PEER REVIEWERS Thomas mack 1, Michael Brodsky 2, Joseph Odumeru 3 1 FDA CFSAN, College Park, MD, USA 2 Brodsky Consultants, Thornhill, Ontario, Canada 3 University of Guelph, Guelph, Ontario, Canada APPLICABILITY OF METHOD Target organism Escherichia coli and Total Coliforms Matrices, raw, frozen raw ground, frozen raw ground and ham REFERENCE METHOD Official Methods of Analysis (2005) 18th Ed., AOAC INTERNATINAL, Gaithersburg, MD, Method : Coiform Group and Escherichia coli. (5) Performance claims MC Media Pad E. coli/coliform sheet medium performed equivalent to the reference method. ORIGINAL CERTIFICATION DATE July 24, 2009 METHOD MODIFICATION RECORD 1. June 2017 Under this AOAC Performance Tested SM License Number, 0901 this method is distributed by: 1. Merck KGaA CERTIFICATION RENEWAL RECORD Renewed Annually through December 2018 SUMMARY OF MODIFICATION 1. Name change to MC Media Pad E. coli/coliform Under this AOAC Performance Tested SM License Number, 0901 this method is distributed as: 1. MC Media Pad E. coli & Coliform Sheet Medium PRINCIPLE OF THE METHOD (1) The MC Media Pad E. coli & Coliform, formerly known as MC Media Pad &Coliform device is a culture medium for the enumeration of total coliform bacteria and differentiation of E. coli. MC Media Pad Aerobic Count and MC Media Pad Coliforms were validated and have been granted AOAC Performance Tested Method Status 3,4). The device consists of a non woven fabric to which a layer of microbial nutrients is deposited on a film. The nutrient film also contains X Gal which is hydrolyzed by beta galactosidase from coliforms to produce a visible blue dye and Salmon glucuronic acid which is hydrolyzed by beta glucuronidase from Escherichia coli to produce a red purple dye. The non woven fabric and nutrient film are constructed on a base film. The basic device (fabric, nutrient and base film) is then inserted between an adhesive backing to stabilize the basic device, and a transparent film. The adhesive backing and the transparent top film serve to deter evaporation, and facilitate inspection of the inoculated device. Sample solutions containing bacteria are deposited on the fabric portion of the device. Then, the sample solution diffuses throughout the entire pad to dissolve and release the nutrient compound film forming a highly viscous solution. Bacteria migrate to the surface of the fabric where they convert the substrates to produce a visible dye after incubation. Escherichia coli produces a purple to indigo spot by the hydrolysis of X Gal and Salmon glucuronic acid, and Coliforms other than E. coli produce green to blue spots by the hydrolysis of X Gal.

3 DISCUSSION OF THE VALIDATION STUDY (1) In the comparison study of MC Media Pad sheet and the reference method using stomacher homogenization, all 100 samples showed no significant difference between the counts of MC Media Pad sheet and the AOAC method In the average count and SDr of 5 subsamples, most of the samples showed no significant difference between both methods. However, in the coliform and E. coli counts of middle and high level inoculated frozen and high level inoculated frozen, the higher counts of sheet might have been due to the difference in growth of the frozen injured bacteria similar to the case of MC Media Pad Coliforms 4). In the case of high and middle level inocurated samples, the results of MC Media Pad sheet reflected the inoculated level than the results of AOAC Method, including in the case of high level inoculated raw. In the independent study, the higher counts of MC Media Pad sheet in the low level inoculated raw might have been caused by the presence of beta galactosidase positive but non gas producing bacteria such as Serratia. In the comparison study of MC Media Pad sheet and the reference method using blender homogenization, 99 out of 100 samples showed no significant difference between the counts of MC Media Pad sheet and the AOAC method , but the count of total coliforms of MC Media Pad in one sample of uninoculated raw was showed significantly higher than that of the AOAC method In the average count and SDr of 5 subsamples, most of the samples showed no significant difference between both methods. However, in the coliform counts of uninoculated raw, the higher coliform counts of MC Media Pad sheet is attributed to the presence of beta galactosidase positive but non gas producing bacteria such as Serratia. In the case of frozen and frozen, the higher E. coli or coliform counts of MC Media Pad sheet might have been due to the difference in growth of the frozen injured bacteria similar to the case of MC Media Pad Coliforms 4) same as the stomacher homogenization study. In the case of high level inoculated raw and raw, the average E coli count of MC Media Pad high level inoculated samples was about 10 times the middle level inoculated samples, but the average count of the high level samples of the AOAC method was 3 to 3.5 times that of the middle level samples. The results of MC Media Pad sheet reflected the inoculated level. The same phenomenon was observed in high level inoculated frozen and frozen. The SDrs of MC Media Pad sheets in both homogenization study were similar or lower as compared to the AOAC method The sheet method was shown to have equal or better repeatability in comparison with the AOAC method. We recognized positive reactions on Serratia and Aeromonas hydrophila grown on MC Media Pad sheets in the exclusivity test, as shown in Table 3 b. These strains are beta galactosidase positive and gram negative rod, and detected as the coliform positive strains by the chromogenic media using the substrate for betagalactosidase. Though we did not recognize gas production, often gas production with Serratia marcescens and Serratia rubidaea in BGLB was reported 11). Serratia marcescens and Serratia rubidaea are opportunistic infection bacteria and A. hydrophila is associated with human gastrointestinal diseases 12 13) Therefore, it might be suitable that these bacteria were detected as a coliform. Table 1 a.comparison between MC Media Pad &Coliform sheet medium and AOAC method for the enumeration of the E. coli and coliform in meat and meat products using stomacher homogenization. (1) Food Inoculated Level coliform E. coli strain (cfu/g) MC Media Pad MC Media Pad Log 10 cfu SDr Log 10 MPN Log 10 cfu SDr Log 10 MPN Confidence limits (P<0.05) Confidence limits (P<0.05) No inoculation <0.98 <0.98 No inoculation <0.98 <0.98 No inoculation <0.98 <0.98 No inoculation <0.98 <0.98 No inoculation <0.98 < >3.04 >

4 > >3.04 > >4.04 > No inoculation <0.98 No inoculation <0.98 No inoculation <0.98 No inoculation <0.98 No inoculation <

5 >3.04 > No inoculation No inoculation <0.98 <0.98 No inoculation <0.98 <0.98 No inoculation <0.98 <0.98 No inoculation <0.98 <

6

7 00 No inoculation <0.98 No inoculation <0.98 No inoculation < No inoculation < No inoculation <

8 No inoculation < No inoculation <0.98 <0.98 No inoculation <0.98 <0.98 No inoculation <0.98 <0.98 No inoculation <0.98 < >3.04 > >3.04 > >3.04 >

9 >3.04 > Samples were homogenized with Masticater Type S for 2 min SDr : Relative standard deviation=(standard deviation of array X)/ (average of array X). Table 3 a. Inclusivity (1) Strains Number of strains BGLB gas production MC Media Pad & Coliforms Strain No or origin Cedecea lapagei 1 + +/blue JCM1684 Citrobacter amalonaticus 1 + +/blue IFO13547 Citrobacter diversus 1 + +/blue JCM1659 Citrobacter freundii 2 + +/blue IFO12681, IID5206 Citrobacter sp /blue From a food Enterobacter aerogenes 2 + +/blue IFO13534, JCM1232 Enterobacter cloacae 2 +/ +/blue IID977, JCM1232 Enterobacter intermedium 1 + +/blue JCM1238 Enterobacter sp /blue From a food Escherichia coli /purple to indigo IID5208, NBRC3301, NBRC3302, NBRC3366, NBRC3543, NBRC3544, NBRC3545, NBRC3546, NBRC3806, NBRC3972, NBRC3991, NBRC12062, NBRC12433, NBRC12734, NBRC13500, NBRC13540, NBRC13891, NBRC13892, NBRC13898, NBRC13966, NBRC14129, NBRC15034, NBRC15484, NBRC Escherichia coli 8 + +/purple to indigo From a food Escherichia fergusonii 1 + +/blue NBRC Escherichia vulneris 1 + +/blue NBRC Klebsiella oxytoca 1 + +/blue JCM1665 Klebsiella planticola 1 + +/blue IFO14939 Klebsiella pneumoniae 2 + +/blue IID5209, JCM1662 Klebsiella sp /blue From a food Kluyvera ascorbata 1 + +/blue JCM1681 Leclercia adecarboxylata 1 + +/blue NBRC Raoultella terrigena 2 + +/blue JCM1687, NBRC : Positive reaction. : Negative reaction.

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11 Table 3 b. Exclusivity (1) Strains Number of strains BGLB gas production MC Media Pad / Coliforms Strain No or origin Aeromonas hydrophila 1 +/blue JCM1027 Bacillus cereus 2 IFO3836, IFO13494 Bacillus megaterium 1 ATCC9885 Bacillus subtilis 2 ATCC6633, IFO3134 Corynebacterium pyrogenes 1 IID547 Corynebacterium xerosis 1 IFO12684 Edwardsiella tarda 1 JCM1656 Enterobacter gergoviae 1 JCM1234 Enterococcus hirae 1 IFO3181 Hafnia alvei 1 JCM1666 Lactobacillus acidophilus 1 JCM1132 Lactobacillus delbruedkii 1 IFO3376 Leuconostoc mesenteroides 1 IFO3832 Micrococcus luteus 2 IFO128, IID862 Morganella morganii 1 IFO3848 Proteus mirabilis 1 IFO13300 Proteus vulgaris 2 IFO3851, IID847 Providencia alcalifaciens 1 IFO12931 Pseudomonas aeruginosa 5 IFO3446, IFO3449, IFO3453, IFO12689, IID1002 Pseudomonas fluorescens 1 NBRC15842 Rahnella aquatilis 1 IFO13544 Rhodococcus equi 1 IFO1311 Salmonella enterica 8 IFO3163, IID1000, JCM1652, NBRC3313, NBRC12529, NBRC13245, NBRC15182, NBRC Salmonella enterica 1 From a chicken Salmonella sp. 3 From a food Serratia marcescens 1 +/blue JCM1239 Serratia rubidaea 1 +/blue IFO12973 Staphylococcus aureus 7 IFO13276, IID5220, JCM2151, JCM2719, JCM2413, JCM2874, NBRC15035 Staphylococcus epidermidis. 1 IFO13889 Staphylococcus hyicus 1 JCM2423 Streptococcus sp. 1 IFO12546 Yersinia aldovae 1 JCM5892 β galactosidase positive gram positive 10 From a food bacteria +: Positive reaction. : Negative reaction. REFERENCES CITED 1. Ushiyama, M. and Iwasaki, M., Evaluation of the Sanita kun E. coli & Coliform Sheet Medium, AOAC Performance Tested SM certification number AOAC Research Institute Validation Outline for Sanita kun E. coli & Coliform Sheet Medium, Approved July H. Morita, M. Ushiyama, S. Aoyama and M Iwasaki, Sensitivity and Specificity of the MC Media Pad Aerobic Count: Internal Validation and Independent Laboratory Study, J AOAC international, 86, (2003) 4. H. Morita, M. Ushiyama, S. Aoyama and M. Iwasaki: Evaluation of the MC Media Pad Coliforms, a dehydrated medium sheet for coliform detection, J. AOAC International, 89, (2006). 5. Official Methods of Analysis (2005) 18th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, Method : Coliform Group and Escherichia coli. 6. FDA Bacterial Analytical Manual online, Chapter 4, Enumeration of Escherichia coli and the Coliform Bacteria, 4.html. 7. Food Hygiene Inspection Guideline, Bacterial ed. p (2004) edited by Japan Food Hygiene Association (in Japanese). 8. M. Manafi, New Development in Chromogenic and Fluorogenic Culture Media, International J. Food Microbiol., 60, (2000). 9. M. Killian and P. Bulow, Rapid Diagnosis of Enterobacteriaceae, Acta Pathol. Scand., Sect B 84, (1976) 10. L. J. Moberg, Fluorogenic Assay for Rapid Detection of Escherichia coli in Food, Appl. Environ. Microbiol., 50, (1985). 11. Bergey s Manual of Determinative Bacteriology, 9th Ed., J.H. Holt et al (Eds), Williams & Wilkins, Baltimore, MD, pp J. L. Smith and P. M. Frantamico, Factors Involved in the Emergence and Persistence of Food borne Diseases, J. Food Prot., 58, (1995) 13. J. H. Isonhood and M. Drake, Aeromonas Species in Foods, J. Food Prot., 65, (2002)