AOAC-RI ERP Book MICRO.pdf

CONFIDENTIAL INFORMATION

Page 3 of 17

speed for 10 min in a Model 16K Microcentrifuge (Bio-Rad Laboratories, Inc.,

Hercules, CA). The supernatant of each centrifuged aliquot was removed and the

pelleted cells were re-

suspended in Butterfield’s Phosphate Buffer (BPB; Made In

House From Various Ingredients). The cells re-suspended in BPB were centrifuged

a second time, and the supernatant was removed once again. The pelleted cells

were re-suspended once more in BPB. These final solutions of cells re-suspended

in BPB were adjusted to a concentration of ~8.00 log

CFU/ml based on the

transmittan

ce of the suspension as determined using a Spectronic™ 200

Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA). This

suspension served as inoculum for

E. coli

O157:H7 and the concentrations of cells

in each were verified on each day of the experiment by serially diluting in BPB and

plating onto Sorbitol MacConkey Agar (SMAC).

2.3 Inoculation of Food Samples

After the

E. coli

O157:H7 culture was prepared, the two different food matrices

were prepared in individual portions of 25 g and 200 g for sprouts and spinach

matrices, respectively. Three portion types were prepared for each testing

method, for each matrix. Each individual portion was placed into a sterile Whirl-

Pak bag. This included a high inoculum level (Portion 1), a low inoculum level

(Portion 2) and un-inoculated samples (Portion 3). After inoculation, all samples

for each inoculum level were removed from each Whirl-Pak bag, combined and

homogenized in one large batch unit and re-aliquoted into the appropriate portion.

In sprouts and spinach, a 7.5 and 0.75 CFU/sample was targeted for the high and

low inoculum levels, respectively. As stated in Section 2.2, the inoculum

concentration was enumerated at the time of inoculation. Minimum volumes

consisting of 300 g (sprouts) and 555 g (spinach) were also removed from each

food matrix and each inoculated portion (i.e. Portion 1 and Portion 2) in order to

perform a five tube-three level Most Probable Number (MPN) analysis on each

portion/matrix as described in Section 2.6. After inoculation, the food samples

were stored at 4°C for 48 h to stabilize. Once this stabilization period was

complete, testing commenced. For each food matrix, a total of 20 samples were

prepared for Portion 2, 5 samples were prepared for Portion 1 and 5 samples were

prepared for Portion 3.

2.4 Analysis of Reference Method Sub-Portions

After the 48 h stabilization period post-inoculation, the samples were subjected to

the corresponding reference method according to the FDA BAM testing

procedures (2).