ERP Micro December 2019

immersed in media. Stomach or homogenize for 2 min or mix well by hand. Incubate for 8 – 16 h at 42 ±1°C.Aseptically weigh 25 g sample into 225 mL prewarmed (42 ± 1°C) mEHEC, C ( a ). Homogenize samples well with masticator, B ( n ). Incubate samples for 6.5–18 h at 42 ± 1°C. For sprout irrigation water, incubate for 8–18 h. (b) Aseptically weigh 375 g sample into 1500 mL prewarmed (42 ± 1°C) mEHEC. Homogenize samples well with masticator. Incubate samples for 8–18 h at 42 ± 1°C.

magnets and tap tips gently to release particles into the Resuspension Buffer Tq. Cover the resuspension plate with adhesive film. E. 10) Repeat steps (6) through (9) for all samples using new tips for each strip of samples. Note : Wear new gloves prior to handling reagents. (a) Mix concentration reagent, C ( b ), in a vortex mixer. Transfer 20 µL to each of the required number of Assurance GDS sample wells (one well/sample), B ( e ), using a repeater pipettor, B ( j ), and 0.5 mL pipet tip, B ( k ). Cover sample wells with adhesive film strips, B ( i ). (b) Transfer 1.0 mL of wash solution, C ( e ), to additional sample wells (one well/sample), using a repeat pipettor and 10 mL pipet tip. Cover sample wells with adhesive film strips. (c) Transfer 45 µL of resuspension buffer, C ( c ), to the sample wells in the resuspension plate, B ( f ), using a repeat pipettor and 0.5 mLpipet tip. Cover resuspension plate with adhesive film strips. (d) From one strip of sample wells, ( a ), carefully remove adhesive film. Using a micropipettor, add 1 mL of incubated sample to each sample well containing concentration reagent. Avoid transferring food particles. A new pipet tip must be used for each sample. Cover each strip of sample wells with a new adhesive film strip prior to adding samples to a new row. Immediately return samples to incubator. (e) Place sealed sample wells held in the sample wells base atop the vortex mixer, B ( d ), and vortex at approximately 900 rpm for 5–15 min. If necessary, adjust vortex speed to avoid liquid contact with adhesive film. After vortexing is completed, remove sample wells base from vortex mixer.

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Note:

Contact Technical Services for recommended

procedures for testing alternate sample sizes.

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F. Sample Preparation Protocol

Note: Sample prep can also be completed using the Assurance ® GDS PickPen TM PIPETMAX ® (PPMX). For automation setup procedures please see the Assurance ® GDS PPMX User Manual (Cat. No. 55240BC).

(a) Sample Preparation Procedure

Note : Change gloves prior to handling reagents.

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1) Aliquot 20 µ L, using a 0.5 mL pipette tip and the repeater pipette, of homogenized O157 Concentration Reagent into the Assurance ® GDS sample wells (1 well/sample). Cover sample wells with adhesive film strips. 2) Add 1.0 mL, using a 10 mL pipette tip and the repeater pipette, of Wash Solution into another set of Assurance ® GDS sample wells (1 well/sample). Cover sample wells with adhesive film strips. 3) Add 45 µ L of Resuspension Buffer Tq, using a 0.5 mL pipette tip and repeater pipette, to the required number of wells in the Resuspension plate. Cover resuspension plate with adhesive film strips. 4) Carefully remove adhesive film strip from 1 strip of sample wells. Remove enriched sample after incubation. Add 1.0 mL of the enriched sample to each sample well that contains the Concentration Reagent. Avoid transferring food particles. A new pipette tip must be used for each sample. Seal each row of the sample wells with new adhesive film strip. Immediately return samples to 42 ° C incubator. 5) Place the sealed sample wells containing O157 Concentration Reagent on the plate vortex mixer at approximately 900 RPM for 5- 15 min. Note: If necessary, adjust the RPM to be certain that liquid does not contact adhesive film. 6) Carefully remove and discard adhesive film strip from 1 strip of samples. Remove corresponding film strip from sample wells containing the Wash Solution. 7) Load tips onto the PickPen TM device, ensuring that the tips are firmly in place on the PickPen TM tool. Extend the PickPen TM magnets and insert tips into the first strip of sample wells. Stir gently for at least 30 sec while continually moving up and down from the surface to the bottom of the wells. Gently tap the PickPen TM tips against the side of the sample wells to remove excess media droplets. 8) Transfer the PickPen TM tips to corresponding sample wells containing Wash Solution and gently swirl for 5 – 10 sec. Tap the PickPen TM tips against the side of the wells to remove excess Wash Solution droplets. Note: Do not release particles into Wash Solution. 9) Transfer PickPen TM tips to the corresponding row of the prepared resuspension plate. With tips submerged, retract the PickPen TM

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