Microbiology Methods for ERP Review 3-2020

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b. Leave the first well in the strip empty to serve as a “blank” for measuring the absorbance of the substrate. c. Aliquot 0.1 mL of the Negative Control in the second well and 0.1 mL of the Positive Control in the third well. Add 0.1 mL of each heated sample to each consecutive well in the strip. If there are wells left over at the end of a test strip the Positive or Negative Controls may be repeated. d. Incubate the plate (containing the strips) at 37 ± 1°C for 30 ± 5 min. e. Following incubation, aspirate the contents of the wells, removing as much of the liquid as possible. Wash the wells 5–7 times with Washing Buffer ensuring completed filling and emptying of the wells through each wash cycle. Note: The washing technique is critical to assay performance; hence it is recommended to use a microplate washer. f. After completion of the washing steps, add 0.1 mL of Conjugate to each well, except for the blank. Upon completion of the addition of the Conjugate, incubate the plate (containing the strips) at 37 ± 1°C for 30 ± 5 min. g. After incubation, stop the reaction by adding 0.1 mL of Stop Solution to all wells, including the “blank” well. Note: The Stop Solution will cause any blue color in wells to change to yellow. h. Where applicable read the optical densities within 10 min in a microplate reader using a 450 nm filter (do not use reference filter), or on the Dynex DS2 instrument using the “Plate Read Only” setting. Inspect the wells before reading for air bubbles and if present burst with a sterile needle. Zero the reader against the “blank” well before the other wells are read. i. Interpretation: The presence or absence of the pathogen is expressed as optical density (OD 450 ) measurements using the Dynex DS2 instrument plate reader or a microplate reader with a 450 nm filter where a Dynex DS2 instrument is unavailable. For control assay acceptance criteria, a result of an OD 450 < 0.100 is valid for a Negative Control, whereas a result of an OD 450 > 0.500 is valid for a Positive Control. A result of an OD 450 < 0.200 is negative for Salmonella . A result of an OD 450 ≥ 0.200 is considered presumptive positive for Salmonella . 6) Solus One Test Result Confirmation a. Assay positive samples may be confirmed by cultural confirmation following the FDA/BAM Chapter 5, USDA/FSIS MLG 4.10 or ISO 6579-1. b. Assay positive samples may also be confirmed by a direct streak to streak from the primary enrichment onto XLD and BSA, in addition a transfer to a secondary enrichment broth may be performed.

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