Microbiology Methods for ERP Review 3-2020

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Washing Buffer bottle (10 mL) to 240 mL of deionized water to prepare the washing buffer at assay 1 concentration. Mix until the activator has fully dissolved. 2 ( c ) Leave the first well in the strip empty to serve as a “blank” for measuring the absorbance of 3 the substrate. 4 ( d ) Aliquot 0.1 mL of the Negative Control in the second well and 0.1 mL of the Positive Control in 5 the third well. 6 ( e ) Add 0.1 mL of each heated sample to each consecutive well in the strip. If there are wells left 7 over at the end of a test strip the Positive or Negative Controls may be repeated. 8 ( f ) Incubate the plate (containing the strips) at 37 ± 1°C for 30 ± 5 min. 9 ( g ) Following incubation, aspirate the contents of the wells, removing as much of the liquid as 10 possible. Wash the wells 5–7 times with Washing Buffer ensuring completed filling and emptying of the 11 wells through each wash cycle. Note: The washing technique is critical to assay performance; hence it is 12 recommended to use a microplate washer. 13 ( h ) After completion of the washing steps, add 0.1 mL of Conjugate to each well, except for the 14 blank. Upon completion of the addition of the Conjugate, incubate the plate (containing the strips) at 37 15 ± 1°C for 30 ± 5 min. 16 ( i ) Repeat the aspiration and wash cycles. 17 ( j ) After completion of the second aspiration and washing steps, add 0.1 mL of TMB Substrate to 18 each well, including the blank. Upon completion of the addition of the TMB Substrate, incubate the 19 plate (containing the strips) for 30 ± 5 min at room temperature (15–25°C). 20 ( k ) After incubation, stop the reaction by adding 0.1 mL of Stop Solution to all wells, including the 21 “blank” well. Note: The Stop Solution will cause any blue color in wells to change to yellow. 22 ( l ) Where applicable read the optical densities within 10 min in a microplate reader using a 450 23 nm filter (do not use reference filter), or on the Dynex DS2 instrument using the “Plate Read Only” 24 setting. Inspect the wells before reading for air bubbles and if present burst with a sterile needle. Zero 25 the reader against the “blank” well before the other wells are read. 26 27 H. Solus One Salmonella (Automated Sample Preparation Method)

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( a ) Prior to analysis, bring the entire contents of the kit to room temperature (15–25°C) by placing

the kit on a sanitized bench top for 1 h prior to use.

( b ) Determine the number of wells required for the test. Take the required number of strips from 32 the pouch and fit them to the frame provided. Note: Unused strips should be returned to the pouch and 33 stored at 2–8°C.

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I. Analysis

( a )

Prepare equipment

1) Turn on power to the Dynex DS2 instrument, followed by the power to the computer. Open the Matrix software on the computer and enter login information. The Dynex DS2

instrument will perform a self ‐ diagnostic check.

( b ) Create a Run File

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