Total Collaborative Study Protocol_Solus One Salmonella v1 1

[4.44 mL Solus  Salmonella  supplement per 1,000 mL Solus BPW (ISO)] to 37 ± 1°C for at least 2 h prior to  1  its addition to a 375g test portion in the sampling bag. Homogenize by hand massaging for 2 min ± 30 s  2  and incubate at 41.5 ± 1°C for 21–22 h.  3  ( h )  Stainless Steel environmental surface 4  For 4” x 4” surface area, sample surface with sterile sampling sponge device (e.g. World Bioproducts  5  EZ Reach Sponge Sampler with HiCap neutralizing broth or Hygiena equivalent). Follow sampling device  6  manufacturers’ instructions for correct use, storage and transport. Pre‐warm 100 mL of Solus BPW (ISO)  7  Medium supplemented with Solus  Salmonella  supplement [4.44 mL Solus  Salmonella  supplement per  8  1,000 mL Solus BPW (ISO) to 37 ± 1°C for at least 2 h prior to its addition to the sampled sponge in the  9  sampling bag. Homogenize by hand massaging for 2 min ± 30 s and incubate at 41.5 ± 1°C for 16–20 h.  10  ( i )  Plastic environmental surface 11  For 1” x 1” surface area, sample surface with sterile sampling swab (e.g. sponge or foam pre‐ 12  moistened with Letheen broth). Follow sampling device manufacturers’ instructions for correct use,  13  storage and transport. Pre‐warm 10 mL of Solus BPW (ISO) Medium supplemented with Solus  14  Salmonella  supplement [4.44 mL Solus  Salmonella  supplement per 1,000 mL Solus BPW (ISO)] to 37 ±  15  1°C for at least 2 h prior to its addition to the sampled swab in the sampling tube. Homogenize by vortex  16  mixing for 2 min and incubated at 41.5 ± 1°C for 16–20 h.  17  ( j )  Following the incubation, gently agitate each bag or tube and transfer a 1 mL aliquot to a  18  polypropylene test tube. Heat tubes at a temperature of 85–100°C for 15 – 20 min.  19  ( k )  After heat treatment, cool the samples to room temperature (15–25°C). This may be  20  accelerated by placing the test tubes in cold water for 5 min.  21  ( l )  Where appropriate use frit filters with denatured matrices that contain high concentrations of  22  particulates to minimize sample handling errors.  23  ( m ) Keep the remaining non‐heat‐treated samples for verification until ELISA results are obtained.  24  These samples can be stored at 41.5 ± 1°C if the ELISA test is to be carried out within 2 h or at 2–8°C for  25  up to 72 h if not.   30  room temperature (15–25°C). Determine the number of wells required for the test. Take the required  31  number of strips from the pouch and fit them to the framer provided. Note: Unused strips should be  32  returned to the pouch and stored at 2–8°C.  33  ( b ) Prepare the Washing Buffer by adding the contents of a single Washing Buffer Activator  34  sachet and Washing Buffer bottle (60 mL) to 1440 mL or a single Washing Buffer Activator sachet and  35  Washing Buffer bottle (10 mL) to 240 mL of deionized water to prepare the washing buffer at assay  36  concentration. Mix until the activator has fully dissolved.  37  ( c )  Leave the first well in the strip empty to serve as a “blank” for measuring the absorbance of  38  the substrate.  39  ( d ) Aliquot 0.1 mL of the Negative Control in the second well and 0.1 mL of the Positive Control in  40  the third well.  41  ( e ) Add 0.1 mL of each heated sample to each consecutive well in the strip. If there are wells left  42 26  27  28  29  G. Solus One Salmonella (Manual Method)  ( a ) Remove the kit from storage at 2–8°C an hour before use to allow the components to reach 

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