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, tray can be read 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 ³ 0.3 indicates positive result. Positive control should give A ³ 1.0 and negative control should give A <0.2. H. 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, and BS plates from tetrathionate broth, selenite cystine broth, and M broth tubes as described in 967.26B ( see 17.9.02), and typical or suspicious colonies should be identified as in 967.26C ( see 17.9.02), 967.27 ( see 17.9.03), and 967.28 ( see 17.9.07). Reference: JAOAC 71 , 973(1988).

on paper towel several times. ( c ) Completely fill each well with working strength wash solution. ( d ) Repeat ( a )–( c ) 2 more times. ( 5 ) Empty tray according to ( 4 )( a ) and ( b ); then add 0.2 mL reconstituted conjugate to each well. Cover tray and incubate 30 min at 35 ° –37°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 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. This way, accurate readings will be obtained. Read result; then continue incubation until positive control has reached a color at least as colored as Panel 3 on the color card or A ³ 1. ( 8 ) Add 0.02 mL stop solution to each well. Incubation time should be ca 10–20 min. If >30 min has elapsed and A of 1.0 has not been attained, test is invalid. G. Reading Results of tests can be determined ( 1 ) visually or ( 2 ) with microtiter tray reader. ( 1 ) Place tray on white background, and then compare individual test wells with color comparator. Positive control should give strong

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