ERP Micro December 2019

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3.3.3 Serially dilute E. coli O157:H7 culture for enumeration in Butterfield’s phosphate buffered diluent (BPBD). Spread plate dilutions onto TSA-YE for counts. Incubate TSA-YE plates at 37 ± 1°C for 18–24 h. Store serially dilutions of the culture at 5 ±

3°C for 48 – 7224 - 42 h.

3.3.4 Count colonies on TSA-YE plates to determine CFU/mL in serial dilution BPBD

tubes.

3.3.5 While BPBD cultures stored refrigerated, offsite customer will scrub surface of beef trim (FSIS Directive 10,010.1 Rev. 4, ( 45 ) ) using Fremonta MicroTally TM MSD cloths. This scrub will adhere typical levels of microflora onto the surface of the MSD cloths. Surface-swabbed cloths will be placed individually into a sterile whirl- pac containers and shipped to testing laboratory overnight delivery at 5 ± 3°. 3.3.6 Inoculate cloth surfaces with test strain as indicated in Table 23 . Low inoculation level will be at partial recovery level (approximately 1 CFU). Inoculation levels will be indicated in report. Allow organism to stabilize on cloth for minimum 12412 h at 5 ± 3°C. Test strains will be cold-stressed (step 3. 43 .3) before

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inoculation onto the cloths.

3. 34 . 73 Begin analyses for each method (candidate and reference) on the same day as

inoculation of the cloths.

3. 4.43.8

GDS O157 Method

3. 43 . 84 .1

Add 200 mL of mEHEC broth to cloth in whirl-pac bag container. If cloth is split, reduce media volume to corresponding smaller cloth size

.

ratio. Homogenize sample by hand or macerate for 2 min.

3. 34 . 84 .2 3. 34 . 84 .3 3. 1.2 9.6.

Incubate for 6.58 – 18 16 h at 41–43°C.

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Complete GDS O157 testing as described in Sections 3. 12 .9.4–

3. 4.53.9

MLG 5.095C.00

3. 43 . 95 .1

Add 200 ± 5 mL mTSB and pummel, blend, or hand massage until

well mixed. Incubate at 42 ± 1°C for 15–24 h.

3. 34 . 59 . 23

Complete MLG 5.095C.00 testing as described in Sections

3. 12 .10.2–3. 12 .10.7.

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3.4 Amplification Tube Consistency Study with PPMX

There are two amplification tube size formats, with an exterior tube volumes of 0.2 mL and 0.1 mL. The contents of the lyophilized primer/polymerase in each tube size are exactly the same (only the exterior tube size is different). This study will examine two lots of GDS O157 kits of each amplification tube size format for product consistency when used with PPMX. Data

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