AOAC ERP MICRO AUGUST 2018

blue-green color indicating that all reagents are functional. If positive control is lighter than “Positive Control” on color comparator card, test is invalid; refer to “Troubleshooting Guide” in package insert, B ( k ). If negative control is darker than “Negative” on color comparator card, it is probable that tray was inadequately washed, and assay must be repeated. ( 2 ) Maximum of blue-green end product occurs at 414 nm. Therefore, read tray at 414 ± 10 nm. For single wavelength readers, set reader to zero (blank) on well containing 200 m L substrate or H 2 O. For dual wavelength readers, set reader to zero (blank) on air and set second reference wavelength at 490 ± 10 nm. A $ 0.3 indicates positive result. Positive control should give A $ 1.0 and negative control should give A <0.2. I. Confirmation of Positive EIA Test Portions Positive EIA reading indicates that Salmonella may be present. However, because antibodies may cross-react with a few other organisms, culture confirmations must be performed by streaking HE , XLD , a nd BS p l a t e s f r om t e t r a t h i on a t e b r o t h , Rappaport-Vassiliadis R10 broth, and M broth tubes as in 967.26 B ( see 17.9.02), and typical or suspicious colonies should be identified as in 967.26 C ( see 17.9.02), 967.27 ( see 17.9.03), and 967.28 ( see 17.9.07). References: J. AOAC Int. 82, 634(1999) ; 87, 374(2004) .

( 5 ) Empty tray according to ( 4 )( a ) and ( b ); then add 0.2 mL reconstituted conjugate to each well. Cover tray and incubate 30min at 36 ° ± 1 ° C. ( 6 ) Empty contents of tray and wash it thoroughly 4 times according to ( 4 )( a )–( c ); then empty tray according to ( 4 )( a ) and ( b ). ( 7 ) Add 0.2 mL reconstituted substrate to each well. Incubate at least 10 min at room temperature (20 ° –25 ° C) until positive control has reached color equivalent to “Positive Control” on color comparator card or to absorbance ( A ) ³ 1.0. Because color development tends to concentrate around edges of wells, it is important to tap sides of plate gently to mix contents prior to reading result. ( 8 ) Add 0.02 mL stop solution to each well. Incubation time should be 10 ! 20 min. If >30 min has elapsed and A of 1.0 has not been attained, test is invalid. H. Reading Positive results may be read ( 1 ) visually with color comparator card where positive result is valid when negative and positive controls match those described on card, or ( 2 ) instrumentally, using filter photometer, where positive result is valid only when negative and positive controls possess acceptable absorbance readings. ( 1 ) Place tray on white background, and then compare individual test wells with color comparator. Positive control should give strong

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