2011 UDM/OECD Technical Workshop

Procedural Revisions to the AOAC Use-dilution Method 

Recommended Action Items and Timeline

Activity Type of Method 

Revision Format

MLB Draft 

Completion

EPA Internal 

Review 

Completion

Anticipated 

Decision by AOAC

Maximum carrier 

counts

Procedural JAOAC 

Manuscript Complete Complete 01/31/2012

Collaborative data and 

use of frozen stocks Procedural JAOAC Manuscript

Draft underway

12/30/2011

01/31/2012 03/31/2012

Reassessment of the 

performance standard –

the statistical model 

and outcome

Procedural JAOAC 

Manuscript

Draft underway

01/31/2011 02/29/2012 04/30/2012

Frozen stocks plus 

reducing daily 

subcultures from 3 to 1

Procedural Written request to 

AOAC

01/31/2011 02/29/2012 04/30/2012

Change 20 mL to 10 

mL final test cultures Procedural Written request to 

AOAC

01/31/2011 02/29/2012 04/30/2012
2011 UDM/OECD Technical Workshop

Editorial Revisions to the AOAC Use-dilution Method 

Recommended Action Items and Timeline

Activity Type of Method 

Revision Format

MLB Draft 

Completion

EPA Internal 

Review 

Completion

Anticipated 

Decision by 

AOAC

Establish maximum time 

carriers remain in 

primary and hold at room 

temp

Editorial Written request to 

AOAC

12/30/2011 1/31/2012 03/13/2012

Clarify step for swirling 

of carrier in the 

disinfectant

Editorial/video Written request to 

AOAC

12/30/2011 1/31/2012 03/13/2012

Clarify step for draining 

of carrier/avoid upper 

part of tube while 

tapping

Editorial/video Written request to 

AOAC

12/30/2011 1/31/2012 03/13/2012

Broth culture media –

use of SB for Sa and Pa 

(for new registrations) 

Editorial Written request to 

AOAC

12/30/2011 1/31/2012 03/13/2012

Timeframe for 

performing control 

counts (3 + 3)

Editorial Written request to 

AOAC

12/30/2011 1/31/2012 03/13/2012
2011 UDM/OECD Technical Workshop

3

Editorial Revisions to the AOAC Use-dilution Method 

Recommended Action Items and Timeline

Activity Type of Method 

Revision Format

MLB Draft 

Completion

EPA Internal 

Review 

Completion

Anticipated 

Decision by 

AOAC

Eliminate use of negative 

tubes for neutralization Editorial Written request to 

AOAC 12/30/2011 1/31/2012 03/13/2012

Add note for recording results 

for subculture media not 

showing turbidity (DE) and 

streak isolation for 

confirmation 

Editorial Written request to 

AOAC 12/30/2011 1/31/2012 03/13/2012

Inspection of P.a. broth 

cultures and add vacuum 

removal; avoid collection of 

bottom layer 

Editorial/pictures Written request to 

AOAC 12/30/2011 1/31/2012 03/13/2012

Under additional guidance, 

use of larger volumes of 

neutralizer if 

primary/secondary 

combination is not effective

Editorial Written request to 

AOAC 12/30/2011 1/31/2012 03/13/2012

Include step for draining 

inoculum from tube with 

carriers prior to removal 

Editorial Written request to 

AOAC 12/30/2011 1/31/2012 03/13/2012
2011 UDM/OECD Technical Workshop

Revisions to MLB SOP MB-05 (Use-dilution Method) 

Recommended Action Items and Timeline

Activity Type of Method 

Revision Format

MLB Draft 

Completion

EPA Internal 

Review 

Completion

Anticipated 

Decision by 

AOAC

Use secondary 

subculture tubes 

on an “as-need 

basis”

Revision to MLB 

SOP MB-05 SOP Revision 12/01/2011 12/31/2012 N/A

Evaluation of 

primary only and 

primary/secondary 

combinations 

Revision to MLB 

SOP MB-17

SOP Revision 12/01/2011 12/31/2012 N/A
2011 UDM/OECD Technical Workshop

OECD Method Issues

• Source(s) and specs for 

carriers

• Filter or plating...

– All treated and controls to 

be filtered

– Options for one versus the 

other

• When to vortex...

– Protocol does not state!

– Briefly after 

neutralization...

– Then 30 sec

• Performance Standards

• Vortex for neutralization 

assay should be the same 

as the test – 30 sec

• Inoculation of additional 

carriers – runoff can be an 

issue

• Use of a positive 

displacement pipet for the 

disinfectant too

• Use of OD for monitoring 

titer of cultures

• What about bubbles? 

5