CERTIFICATION. Certificate No. The AOAC Research Institute hereby certifies that the performance of the test kit known as: Medi Ca SA.
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1 CERTIFICATION AOAC Performane Tested SM Certifiate No The AOAC Researh Institute hereby ertifies that the performane of the test kit known as: Medi Ca SA manufatured by Dai Nippon Printing Co., Ltd , Ihigaya Kagaho Shinjuku, Tokyo Japan This method has been evaluated in the AOAC Performane Tested Methods SM Program, and found to perform as stated by the manufaturer ontingent to the omments ontained in the manusript. This ertifiate means that an AOAC Certifiation Mark Liense Agreement has been exeuted whih authorizes the manufaturer to display the AOAC Performane Tested SM ertifiation 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 alendar year from the date of this ertifiate (November 15, 2017 Deember 31, 2018). Renewal may be granted at the end of one year under the rules stated in the liensing agreement. Deborah MKenzie Deborah MKenzie, Senior Diretor Signature for AOAC Researh Institute November 15, 2017 Date 2275 Researh Blvd., Ste. 300, Rokville, Maryland, USA Telephone: Fax: Internet e mail: aoari@aoa.org * World Wide Web Site:
2 METHOD AUTHORS Mai Shimizu, Kentaro Takenaka, Takeo Suzuki, Aya Miyasaka, Taiki Matsuda, Tatsuhiko Iwase, and Hitoshi Kyotani SUBMITTING COMPANY Dai Nippon Printing Co., Ltd , Ihigaya Kagaho Shinjuku, Tokyo Japan KIT NAME(S) Medi Ca SA CATALOG NUMBERS SA 01 INDEPENDENT LABORATORY Q Laboratories, In Harrison Avenue Cininnati, OH USA AOAC EXPERTS AND PEER REVIEWERS Yi Chen 1, Yvonne Salfinger 2, Wayne Ziemer 3 1 US FDA CFSAN, College Park, MD, USA 2 Consultant, Denver, CO, USA 3 Consultant, Loganville, GA, USA APPLICABILITY OF METHOD Target organisms Staphyloous Matries raw beef, raw ground beef, raw lamb, ooked ham, raw salmon, frozen prawn, fresh hilled pasta, pasteurized milk, natural heese, ream puff and potato salad REFERENCE METHOD AOAC Offiial Method SM , Staphyloous in Foods, ( (2) Performane laims Performane equivalent to that of the AOAC Offiial Methods SM , Staphyloous in Foods ORIGINAL CERTIFICATION DATE November 15, 2017 METHOD MODIFICATION RECORD NONE Under this AOAC Performane Tested SM Liense Number, this method is distributed by: NONE CERTIFICATION RENEWAL RECORD New Approval through 2018 SUMMARY OF MODIFICATION NONE Under this AOAC Performane Tested SM Liense Number, this method is distributed as: NONE PRINCIPLE OF THE METHOD (1) Medi Ca SA is a ready made dry medium for Staphyloous ount made up of four omponents: a waterproof sheet, a dry medium ontaining a gelling agent and the hromogeni enzyme substrates, a hydrophobi resin ring surrounding the medium, and a transparent over over the medium (Figure 1). Figure 2 shows the priniple of the Medi Ca SA Method. A sample suspension is dispensed on the enter of the medium while the over is lifted. After that, the over is gently dropped to evenly spread the suspension on the medium. The suspension rapidly soaks into the medium, whih turns into a gel in 3 minutes. The inubation of the sheet at 35 ± 1 or 37 ± 1 C for 24 ± 1 h develops blue olonies for S. beause of the enzymati reation involving the substrate. DISCUSSION OF THE VALIDATION STUDY (1) In the exlusivity study, S. gallinarum (NBRC ), B. ereus (NBRC 13494, #D0068) and B. liheniformis (NBRC 12200) formed pink olony. The reason for this was that the type of enzyme produed from these strains was different from S.. Those strains were easily distinguished from S. by olony olor. However, S. shleiferi (ATCC 43808) formed blue olony, whih was the same as S.. In this ase, oagulase test is neessary to identify S. or not. S. shleiferi showed negative oagulase reation. Some of the S. related bateria suh as Staphylooi and Baillus speies grow in Baird Parker medium. When testing the raw food matries, these olonies sometimes make enumeration of S. diffiult. In ase of Medi Ca SA method, the growth of these bateria is inhibited and S. olony is stained learly. Medi Ca SA method does not need to onfirm halo of olony. In the matrix study, all matries were inubated at two temperatures, 35 or 37 ± 1 C. It was observed that the olony intensity was stronger at 37 C than 35 C for some of the matries. However, the number of olonies were almost the same at these two temperatures, and there were no signifiant differenes. Overall, it was generally observed that the Medi Ca SA method produed statistially similar results when ompared to the referene method. This rapid method makes it possible to simultaneously detet and enumerate S. in only 24 hours, while the referene method requires 48 hours.
3 Dai Nippon Medi SA AOAC Certifiation Number Table 1. Inlusivity Study (1) Strain Name Soure Origin Medi Ca SA a 1 Staphyloous ATCC 6538 b Human lesion Blue 2 Staphyloous ATCC 8095 Cream pie Blue 3 Staphyloous ATCC 9144 Unknown Blue 4 Staphyloous ATCC Ham Blue 5 Staphyloous ATCC Unknown Blue 6 Staphyloous ATCC Clinial isolate Blue 7 Staphyloous ATCC Chiken tetrazzini Blue 8 Staphyloous ATCC Unknown Blue 9 Staphyloous NBRC Unknown Blue 10 Staphyloous NBRC Human lesion Blue 11 Staphyloous NBRC Wound Blue 12 Staphyloous NBRC Human pleural fluid Blue 13 Staphyloous NCTC d Human lesion Blue 14 Staphyloous #D0072 e Ground beef and pork Blue 15 Staphyloous #D0075 Chiken Blue 16 Staphyloous #D0076 Chiken Blue 17 Staphyloous #D0088 Ground pork Blue 18 Staphyloous #D0106 Human skin Blue 19 Staphyloous #D0107 Human skin Blue 20 Staphyloous #D0108 Human skin Blue 21 Staphyloous #D0109 Human skin Blue 22 Staphyloous #D0112 Food poisoning Blue 23 Staphyloous #D0113 Food poisoning Blue 24 Staphyloous #D0116 Food poisoning Blue 25 Staphyloous #D0117 Food poisoning Blue 26 Staphyloous #D0118 Food poisoning Blue 27 Staphyloous #D0120 Food poisoning Blue 28 Staphyloous #D0121 Food poisoning Blue 29 Staphyloous #D0124 Food poisoning Blue 30 Staphyloous #D0125 Food poisoning Blue 31 Staphyloous #D0130 Food poisoning Blue 32 Staphyloous #D0131 Food poisoning Blue 33 Staphyloous #D0133 Food poisoning Blue 34 Staphyloous #D0134 Food poisoning Blue 35 Staphyloous #D0135 Food poisoning Blue 36 Staphyloous #D0138 Food poisoning Blue 37 Staphyloous #D0151 Milk Blue 38 Staphyloous #D0152 Milk Blue 39 Staphyloous #D0153 Milk Blue 40 Staphyloous #D0154 Milk Blue 41 Staphyloous #D0156 Milk Blue 42 Staphyloous #D0182 Ground pork Blue 43 Staphyloous #D0183 Ground pork Blue 44 Staphyloous #D0185 Chiken Blue 45 Staphyloous #D0206 Unknown Blue 46 Staphyloous #D0207 Unknown Blue 47 Staphyloous #D0208 Pork Blue 48 Staphyloous #D0209 Pork Blue 49 Staphyloous #D0210 Pork Blue 50 Staphyloous #D0211 Pork Blue 51 Staphyloous #D0216 Food Blue 52 Staphyloous #D0217 Food Blue a Colony olor. b Amerian Type Culture Colletion, Manassas, VA. Biologial Resoure Center, National Institute of Tehnology and Evaluation, Chiba, Japan. d National Colletion of Type Cultures, a Culture Colletion of Publi Health England, Salisbury, UK e Numbers starting with #D indiates strains that were isolated by Dai Nippon Printing Co., Ltd.
4 Dai Nippon Medi SA AOAC Certifiation Number Table 2. Exlusivity Study (1) Strain Name Soure Origin Medi Ca SA a 1 Staphyloous auriularis ATCC b External auditory meatus 2 Staphyloous apitis ATCC Human skin 3 Staphyloous aprae ATCC Goat milk 4 Staphyloous arnosus NBRC Dry sausage 5 Staphyloous arnosus #D0086 d Roast beef 6 Staphyloous ohnii NBRC Human skin 7 Staphyloous epidermidis NBRC Unknown 8 Staphyloous epidermidis NBRC Nose 9 Staphyloous gallinarum NBRC Chiken nares Pink 10 Staphyloous sp. #D0058 Ground beef and pork 11 Staphyloous haemolytius NBRC Human skin 12 Staphyloous hominis ATCC Blood 13 Staphyloous hyius ATCC Pig with exudative epidermitis 14 Staphyloous intermedius ATCC Pigeon nares 15 Staphyloous lentus ATCC Goat udder 16 Staphyloous saprophytius NBRC Urine 17 Staphyloous shleiferi ATCC Jugular atheter Blue 18 Staphyloous siuri ATCC Eastern gray squirrel skin 19 Staphyloous simulans NBRC Human skin 20 Staphyloous warneri NBRC Human skin 21 Staphyloous xylosus NBRC Human skin 22 Baillus irulans NBRC Soil 23 Baillus ereus NBRC 3836 Unknown 24 Baillus ereus NBRC Unknown 25 Baillus ereus NBRC Unknown Pink 26 Baillus ereus #D0068 Food powder Pink 27 Baillus liheniformis NBRC Unknown Pink 28 Baillus subtilis NBRC 3134 Unknown 29 Baillus thuringiensis NBRC 3951 Unknown 30 Baillus pumilus NBRC Unknown 31 Enteroous faealis NBRC Unknown 32 Enteroous faealis ATCC Urine 33 Enteroous faeium NBRC Unknown 34 Leuonosto mesenteroides NBRC 3426 Unknown 35 Maroous aseolytius ATCC Dairy produts 36 Maroous aseolytius #D0073 Ground beef 37 Miroous luteus NBRC 3333 Unknown 38 Aeromonas hydrophila NBRC Unknown 39 Citrobater freundii ATCC 8090 Unknown 40 Enterobater aerogenes NBRC Sputum 41 Enterobater loaae NBRC Spinal fluid 42 Esherihia oli NBRC 3972 Fees 43 Esherihia oli NBRC Urine 44 Esherihia oli ATCC Clinial isolate 45 Klebsiella oxytoa NBRC Pharyngeal tonsil 46 Klebsiella pneumoniae ATCC Unknown 47 Kluyvera ryoresens NBRC Kithen food 48 Proteus mirabilis NBRC Unknown 49 Pseudomonas aeruginosa NBRC 3899 Well water 50 Pseudomonas aeruginosa ATCC 9027 Outer ear infetion 51 Salmonella enteria NBRC Human fees 52 Serratia maresens NBRC Pond water 53 Aspergillus niger NBRC Tannin galli aid fermentation 54 Candida albians NBRC 1594 Clinial bronhomyosis 55 Saharomyes erevisiae NBRC Brewer's top yeast a Colony olor, = not deteted. b Amerian Type Culture Colletion, Manassas, VA. Biologial Resoure Center, National Institute of Tehnology and Evaluation. d Numbers starting with #D indiates strains that were isolated by Dai Nippon Printing Co., Ltd.
5 Table 3. Matrix Study: Medi Ca SA vs. AOAC C (1) 35 C Matrix Inoulation Contamination Medi Ca SA Baird Parker Mean 95% CI d Mioorganism Level Mean a b s r RSD r Mean s r RSD r Differene LCL e UCL f r 2g Cooked ham Uninoulated <1.00 <1.00 Staphyloous Low Medium D0109 High Cream puff Uninoulated <1.00 <1.00 Staphyloous Low Medium ATCC 8095 High Fresh hilled pasta Uninoulated <1.00 <1.00 Staphyloous Low Medium NBRC High Frozen prawn Uninoulated <1.00 <1.00 Staphyloous Low Medium NBRC High Natural heese Uninoulated <1.00 <1.00 Staphyloous Low Medium ATCC High Natural heese h Uninoulated <1.00 <1.00 Staphyloous Low Medium ATCC High Pasteurized milk Uninoulated <1.00 <1.00 Staphyloous Low Medium D0152 High Potato salad Uninoulated <1.00 <1.00 Staphyloous Low Medium D0138 High Raw beef Uninoulated <1.00 <1.00 Staphyloous Low Medium NBRC High Raw beef h Uninoulated <1.00 <1.00 Staphyloous Low Medium ATCC High Raw ground beef h Uninoulated <1.00 <1.00 Staphyloous Low Medium ATCC High Raw lamb Staphyloous Uninoulated <1.00 <1.00 Low
6 Dai Nippon Medi SA AOAC Certifiation Number ATCC Medium High Raw salmon Uninoulated <1.00 <1.00 Staphyloous Low Medium D0076 High a Mean of 5 repliates after the logarithmi transformation: Log 10 [CFU/g + (0.1)f]. b s r = standard deviation. RSD r = relative standard deviation. d CI = onfidene interval. e LCL = lower onfidene limit. f UCL = upper onfidene limit. g r 2 = square of the orrelation oeffiient. h Matrix study onduted by the independent laboratory. Table 4. Matrix Study: Medi Ca SA vs. AOAC C (1) 37 C Matrix Inoulation Contamination Medi Ca SA Baird Parker Mean 95% CI d Mioorganism Level Mean a b s r RSD r Mean s r RSD r Differene LCL e UCL f r 2g Cooked ham Uninoulated <1.00 <1.00 Staphyloous Low Medium D0109 High Cream puff Uninoulated <1.00 <1.00 Staphyloous Low Medium ATCC 8095 High Fresh hilled pasta Uninoulated <1.00 <1.00 Staphyloous Low Medium NBRC High Frozen prawn Uninoulated <1.00 <1.00 Staphyloous Low Medium NBRC High Natural heese Uninoulated <1.00 <1.00 Staphyloous Low Medium ATCC High Natural heese h Uninoulated <1.00 <1.00 Staphyloous Low Medium ATCC High Pasteurized milk Uninoulated <1.00 <1.00 Staphyloous Low Medium D0152 High Staphyloous Uninoulated <1.00 <1.00 Potato salad Low D0138 Medium
7 Dai Nippon Medi SA AOAC Certifiation Number High Raw beef Uninoulated <1.00 <1.00 Staphyloous Low Medium NBRC High Raw beef h Uninoulated <1.00 <1.00 Staphyloous Low Medium ATCC High Raw ground beef h Uninoulated <1.00 <1.00 Staphyloous Low Medium ATCC High Raw lamb Uninoulated <1.00 <1.00 Staphyloous Low Medium ATCC High Raw salmon Uninoulated <1.00 <1.00 Staphyloous Low Medium D0076 High a Mean of 5 repliates after the logarithmi transformation: Log 10 [CFU/g + (0.1)f]. b s r = standard deviation. RSD r = relative standard deviation. d CI = onfidene interval. e LCL = lower onfidene limit. f UCL = upper onfidene limit. g r 2 = square of the orrelation oeffiient. h Matrix study onduted by the independent laboratory
8 REFERENCES CITED 1. Shimizu, M., Takenaka, K., Suzuki, T., Miyasaka, A., Matsuda, T., Iwase, T., and Kyotani, H., Evaluation of the Dai Nippon Medi SA, AOAC Performane Tested SM ertifiation number AOAC Offiial Method SM , Staphyloous in Foods, ( 3. Bergey s Manual of Systematis of Arhaea and Bateria, Staphyloous, U.S. Food and Drug Administration (2013) FDA Bateriologial Analytial Manual, Chapter 12, ( 5. Offiial Methods of Analysis (2012), 19th Ed., Appendix J, AOAC INTERNATIONAL, Rokville, MD, ( 6. Least Cost Formulations, Ltd., Paired Method Analysis for Miro Testing (2010), Version 1.0, ( method analysis for miro.xlsx) 7. AOAC Researh Institute Performane Tested Methods SM Program validation outline protool: Independent Laboratory Study for the Dai Nippon Printing Company, Ltd. for the Medi Ca SA Medium for Enumeration of Staphyloous (Version 1, February 2017)
CERTIFICATION. Certificate No. The AOAC Research Institute hereby certifies that the performance of the test kit known as: Compact Dry X SA
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