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UDM/OECD Technical Workshop – Highlights from Laboratory Sessions

(November 15th, 2011: Further Standardization of the AOAC UDM)

Method/Activity Aspect Comments/recommendations

UDM

Test Cultures/Growth Media

Nutrient broth (NB) vs. 

synthetic broth (SB)

Both media are specified in the Use Dilution Methods 

(UDMs); EPA has historically used NB for daily and 

final test cultures.

MLB stresses the need to identify a single medium. 

Clumping of S. aureus has been frequently observed by 

the group in NB and the medium is difficult to 

standardize (e.g., beef extract powder vs. paste, boiling 

of media, color of prepared media).

A handout was provided by EPA showing optical 

density and carrier counts of S. aureus grown in nutrient 

broth and synthetic broth. Optical density (OD) and 

counts were similar, regardless of the growth media.

EPA stated that the type of broth culture medium is 

identified in approximately 50% of the regulatory 

jackets.

A participant speculated that using different growth 

media may result in different efficacy testing outcomes.

ACTION ITEM: Consensus on the issue:

EPA will seek an editorial revision to remove 

nutrient broth from the AOAC UDMs; however, for 

ATP efficacy testing, EPA will utilize the medium 

identified in the registration data for currentlyregistered products. For future registrations, 

synthetic broth will be used. AD will communicate 

this point following acceptance by AOAC. 

Regulatory guidance will not be included in the official 

method. 

20 mL vs. 10 mL for test EPA briefly explained the history of the 10 and 20 mL 
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cultures; removal of pellicle 

during harvest

issue – ambiguous language was in the 1990 version of 

the method. EPA SOP MB-05 specifies 20 mL final test 

cultures. Some labs have converted over to 20 mL, but 

most have retained the 10 mL volume.

EPA demonstration: Removal of pellicle of P. 

aeruginosa from 10 mL and 20 mL test culture tubes 

using vacuum and pipette aspiration.

A handout was provided showing titer, optical density, 

and carrier counts for S. aureus and P. aeruginosa

cultures grown in both 10 mL and 20 mL tubes of 

nutrient broth. Titer, optical density, and carrier counts 

were similar, regardless of the volume of growth media. 

The group concurred on a recommendation to use 

vacuum aspiration as the principal method to remove the 

pellicle from 10 mL tubes.

When harvesting either organism, avoid sediment l 

which has settled to the bottom of the tube; add a 

statement to the official UDM.

The group stressed the need to carefully observe the 

final test culture tubes for pellicle fragments prior to 

culture harvest and after pooling of culture.

ACTION ITEM: A recommendation was made to 

include a statement in the official method regarding 

inspection of the Pseudomonas broth cultures for 

evidence of pellicle.

10 mL test culture tubes are more convenient for 

efficient pellicle removal; pellicle in 20 mL cultures was 

less intact, more fragile.

ACTION ITEM: EPA interpretation of method is to 

use 20 mL test culture volumes; however, EPA is 

willing to pursue revision to the official UDMs to 

change the 20 mL specified in the method to a 10 mL 

volume.
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Carrier inoculation

EPA stated that the AOAC UDM is specific – 20 

carriers added to 20 mL of inoculum

One participant stated that there are two ways of 

inoculating carriers: add carriers to a tube of inoculum 

or add inoculum to a tube of carriers.

Some labs remove inoculum at the end of the 15-min 

inoculation period using vacuum aspiration rather than 

removing carriers from the tube.

Complete coverage of the carriers is necessary.

Ratio of carrier to volume of inoculum is important

Appropriate sized vessel must be used – some labs are 

not using the prescribed tube size, rather, are using 

larger vessels while maintaining the 1:1 ratio. Use of a 

larger vessel is a deviation from the method.

Group agreed that removal of the inoculum may aid in 

transferring less liquid on carriers, thus enhancing the 

drying process.

ACTION ITEM: EPA will submit a recommendation 

to include removal (aspiration) of inoculum from the 

tube as an option.

ACTION ITEM: EPA to consider submitting a 

recommendation to include alternate method for use 

of a larger vessel to inoculate carriers as long as 1:1 

carrier to inoculum volume is maintained. This 

proposal may require data generation by industry. 

Carrier transfers

The method currently directs to “briefly swirl” carrier 

upon transferring carrier into the disinfectant. EPA

stated that no swirling of the tube is preferred, i.e., why 

swirl at all? There is a risk of increasing wash-off into 

the disinfectant. However, some members thought that 

swirling was necessary, especially for viscous products 

that may trap air bubbles. 
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Draining the carrier by tapping following the contact 

time is specified. Some labs do not tap at all while 

others drain (drip) outside the tube. Important to tap 

away from neck of tube. 

Avoid upper part of tube while tapping.

ACTION ITEMS: Clarify step for swirling of the 

carrier in the disinfectant and tapping. Submit 

written recommendation and video clip to AOAC. 

Use same technique for neutralization as well. 

Include language to avoid tapping against the upper 

part of the tube to mitigate cross contamination.

UDM

Primary and secondary tubes

EPA stated that neutralization is not conducted for each 

test, but secondary tubes are used.

Secondary tubes can be used on an as-need basis; a 

neutralization assay will need to be conducted for each 

product sample.

If EPA has issues with neutralization even after using 

secondary tubes, EPA will contact the company to 

discuss.

ACTION ITEM: EPA to revise SOP MB-05 to 

incorporate use of secondary tubes on an “as-need 

basis”.

Turbidity 

Growth/no growth

Use of DE broth: Positive results in DE broth are a 

color change (Staph) and presence of pellicle (Pseudo)

can be problematic.

It was suggested to incubate the DE tubes for an 

additional day so the pellicle will be more visible.

Streak all tubes to confirm growth (can be cumbersome)

Several labs streak the negative tubes on to agar media 

to verify negative tubes. EPA to change strategy for 

conducting neutralization confirmation – this will be 

performed prior to each UDM with primary only and 

primary/secondary combinations.
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There was a question on increasing the volume of the 

neutralizer to 20 mL, several people said they did it 

successfully. EPA does not increase the volume of the 

neutralizer as this is a method deviation. Will require 

data generation by industry.

Action Item: EPA will propose the addition of new 

text to AOAC method describing the different 

neutralization/subculture scenarios.

Carrier Counts

Synchronize carrier counts with the product test.

Action Item: 3 carriers will be assayed at the 

beginning of the test and 3 at the end.

Carrier screening

Visual inspection of carriers in necessary; however, the 

importance of bioscreening was raised by EPA. The 

group did not concur with the elimination of 

bioscreening.

UDM/OECD Technical Workshop –Highlights from Laboratory Sessions

(November 16th, 2011: Further Standardization of the OECD Method)

OECD Method

(SOP MB-25)

Carriers

Source of carriers

Several sources of carriers discussed – this may be an 

issue that impacts the outcome of tests. Action item -

CSPA will assist with sources of carriers.

MLB will continue to work with the OECD VMG to 

make a final decision on source(s) and specs prior to 

launch of the collaborative study.

Action Item: Specs will be discussed at the next VMG 

meeting. 

OECD Method

(SOP MB-25) Carrier screening

Visual screening without the aid of magnification seems 

suitable.

Action items: Examples of failed carriers would be 

helpful – EPA to provide. 

OECD Method

(SOP MB-25)

Inoculum preparation

Preparation and 

standardization of the 

inoculum 

The group discussed the possibility of concentrating 

both organisms the same way through centrifugation and 

resuspension. 
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Action item – further discussion with the VMG will be 

required.

OECD Method

(SOP MB-25)

Carrier treatment

Product application

Proposal to add “inoculate extra carriers” due to 

possible runoff – the group concurred. Use of a positive 

displacement pipette for dispensing the product onto 

carriers was discussed; there was general agreement to 

this idea. Vortex time is not specified in the method –

agreed to add “vortex briefly” immediately following 

addition of the neutralizer. 

Action Item: Items will be discussed at the next VMG 

meeting. 

OECD Method

(SOP MB-25) Collaborative study

EPA led a collaborative study to verify the performance 

of the OECD method. Several participants expressed 

interest in this proposal.

Action item – EPA will consult with CSPA for 

volunteers as the collaborative study is developed.

OECD 

Filtration

Group agreed to filter all samples using 0.22 μm filters 

and to eliminate options for plating controls and use of 

0.45 micron filters.

Action Item: Issues will be discussed at the next 

VMG meeting. 

Carrier-based neutralization 

assay

A demonstration was conducted and the issue of % 

difference (75%) for treatment comparison was discussed. 

Research topics with 

industry – pre-collaborative

studies

The group discussed the research priorities associated with

OECD method – impact on the Log Reduction with quat 

products. EPA continues to work with the ADBAC group to 

better understand the impact of growth media, hard water 

and the carrier surface on the low efficacy of quat-based 

products. Minor changes to the OECD method must be 

made before the collaborative study early in 2012. ADBAC 

will provide a set of product samples, EPA has provided 

product samples. Management of Pseudomonas pellicle will 

be an issue if growth medium other than 1:1000 diluted 
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TSB is used. 

OECD Streamlining label claims The group discussed EPA’s goal to test all disinfectants 

with soil, hard water, and a 5 minute contact time.