AOAC RI ERP Final Action Recommendations

SUBMITTED 050113

Media Preparation

Follow instructions provided with media. Twelve plates of each of the 7 media types will be required.

Test Cultures

Each collaborator will receive a set of 12 pure bacterial cultures on agar slants. These will be shipped by

overnight courier from the Study Director’s laboratory with Category B biohazard packaging and

labeling. The cultures will be blind coded. All should be treated as pathogens and Biosafety level 2

precautions employed in their handling. When received, the cultures should be stored at 2-8

C until the

start of testing. The Sample Receipt Form (appended) is to be completed and returned to the Study

Director on the day samples are received.

Study Protocol

Two independent operators will be required at each collaborating laboratory, one to prepare and code

the test samples, and a second to conduct the ANSR analyses and record results.

Specific dates for sample receipt and analysis will be provided by the Study Director.

Detailed Tasks by Day

Day 1 Collaborators receive test cultures

Return completed Sample Receipt Form, provide name and contact information for “Operator

1” for purposes of sample blind coding.

Complete media preparation (agar plates and sterile PBS tubes)

Day 2 Operator 1 – Streak test cultures (12) to agar media (7 types). Streak each coded culture to its

own plate, streaking for isolated colonies. Incubate plates for 24 + 2 hours at 35 + 1

Day 4 Operator 1 – Randomize and code all plates following instructions provided by the Study

Director. Operator 2 is not to participate in or observe this activity.

Operator 1 – From each of the 84 plates, pick an isolated colony and resuspend the colony in 0.5

mL sterile PBS. Vortex or otherwise mix so that the colony is completely resuspended. Arrange

tubes in order 1-84. Operator 2 is not to participate in or observe this activity.

Note: It is anticipated that all test strains will grow on all 7 agar media. In case of any situations

of no growth, skip this sample and its coded number and instruct Operator 2 to note accordingly

on the Data Recording Form.