Method Activity Aspect EPA Suggestion/Recommendation CSPA Comments Priority

SOP MB-09-04 Carrier Innoculation

Size of Petri dishes with and without 

filter paper

EPA’s SOP calls for standard Petri dishes 

without filter paper. Industry is using filter paper 

and some labs are using shallow Petri dishes to 

allow for easier wiping. Action is required on 

standardizing the use of filter paper and shallow 

dishes.

The use of a shallow petri dish would allow the 

testing to be performed in the dish and cut down on 

the handling of the slide. Most labs use filter paper 

already and this might cut down on the movement of 

the slide during wiping. H-3

SOP MB-09-04 Carrier Wiping Position of the glass slide carrier during w

EPA holds carrier on edge of Petri dish while 

industry wipes carrier inside Petri dish. Action 

item – standardization of the position of the 

carrier during wiping is encouraged. EPA’s 

method requires two people while the industry 

approach can be achieved by a single analyst. 

Methods requiring a single analyst are 

encouraged.

The use of a shallow dish would be better so the 

slide does not need to be held. This will also allow 

one person to run the test. H - 1

SOP MB-09-04 Control Carrier Counts Timing of the carrier counts

EPA performs counts during the initial phase of 

the study; however, industry performs counts at 

the end. Vortexed carriers may be refrigerated 

prior to dilution and plating (EPA). Parties 

considered an approach where counts could be 

split, beginning and end, and then averaged. 

Also, the group agreed that if a single carrier fell 

below the established minimum, it was the mean 

value across all carriers analyzed that would be 

used to qualify the study. Action item – seek 

consensus on timing of processing control 

carrier counts.

It is not clear that all aspects of averaging carriers 

have been reviewed as well as how the average 

would be used. If the carriers plated last were below 

the required carrier count number they should not be 

averaged with carriers from the beginning to bring 

the results into compliance. It seems that the 

carriers should be plated at the end of the testing or 

carriers should be done with each set and used for 

the set, not averaged. This will be a good item to 

discuss H - 1

Draft Quantitative Petri 

Plate Method Wiping Wiping and folding

EPA demonstrated the use of 4 folds and the 6 

total revolutions/corkscrew process. The group 

discussed the advantages of folding one time 

(inverted) when reaching the center to provide a 

fresh surface. The group generally agreed with 

this process. Action item – EPA will incorporate 

this step in the draft procedure. 

This method was demonstrated at the ASTM 

meeting showing that the cloth is turned for the 

second set of 3 swirls. The change should be 

confirmed. H – 1 

Draft Quantitative Petri 

Plate Method Wiping Performance standard

Is it appropriate to put the performance criteria 

(regulatory language) in the method? How was 

the Disinfectant level decided and determined to 

be appropriate? CSPA has gone on record with 

AOAC that regulatory language does not belong 

in the method. 

CSPA would like to discuss the options for 

performance criteria being in the method or noted in 

a place which is easily accessible. At the least 

CSPA would like to have a discussion on EPA’s 

thoughts on performance standards for NFC 

sanitizers and disinfectants H - 1

Use-Dilution Method 

(SOP MB-05)

Bioscreening and 

efficacy testing

Efficacy testing (primary and secondary 

subcultures)

EPA consistently uses both primary and 

secondary subculture tubes, industry typically 

uses primary tubes only. Some concerns were 

expressed regarding the need to do both and the 

increased potential for contamination. EPA does 

not perform a neutralization assay with every 

test, thus use of both primary and secondary 

tubes are deemed necessary. The issue of 

increased resources and time was also 

expressed. EPA has not observed increased 

contamination rate with use of secondary tubes. 

No action items. 

This is a big topic of discussion. There is more 

chance for contamination with the use of secondary 

sub-culture tubes. If the jacket for each product is 

available the neutralization media used for 

registration should be used by EPA. H – 1

SOP MB-09-04 And for 

SOP MB (GST) Carrier Inoculation Glass slide carriers

Use of frosted glass microscope slides (inverted) 

common by industry – EPA will likely use them 

in the future as well. No action required.

CSPA highly recommends the use a frosted end 

microscope slide to avoid making any marks on the 

slide to assure no ink is transferred to the product 

during testing. M-H – 2

Use-dilution method 

(SOP MB-05) Culture harvest and carr Carrier exposure

EPA uses Teflon-coated (Pyrex) tubes to reduce 

breakage of tubes during carrier autoclaving. 

EPA does not remove broth prior to placing 

carrier on filter paper for drying. Industry 

suggests removing broth first to mitigate the 

wetness of carriers. Action item – seek revision 

to the official method and EPA’s SOP to include 

this step. 

Test organism should be removed (poured off or 

suctioned off) from the tube with the carriers. 2 

reasons – the carriers will have less liquid retained 

on them ensuring more consistent and even drying - 

soak time will be the same for all carriers.

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 look into sources of 

carriers .

Dr. Syed Sattar states that if the same material is 

used he doesn’t see that there would be an issue. 

This is something that hasn’t been tested. CSPA 

participants discussed that the Test Guidelines 

should indicate that the beveled (smooth edged) 

side should be the consistent side to use for testing. 

This will be discussed with the OECD VMG by Diane 

Falbo and Mary Bennett. OECD doesn’t allow 

sources for equipment to be listed in guidelines. It is 

recommended that a single source for the carriers 

be listed for consistency. M-H - 3

SOP MB-09-04

Container of 

Towelettes Canister/Container of Towelettes

The container of the towelettes is shook prior to 

removing any towelettes. If there are not directions 

on the label then the practice shouldn’t be done. M - 3

SOP MB-09-04 Carrier Wiping Timing of exposure

General agreement that the contact time begins 

following the wiping process; however, it would 

be advisable to indicate this in EPA’s SOP.

As long as the carrier has the allotted time for 

exposure there is no issue. SOP should be 

updated immediately to reflect this information

M – 1, 

their 
SOP MB-09-04 Control Carrier Counts Carrier processing

Some disagreement in methodology exists, i.e., 

EPA vortexes carriers, some in industry sonicate 

then vortex. Action item – seek consensus on 

inoculum removal/disaggregation process.

NOTE: need some discussion on this point to get 

input. Method states vortex or sonicating. How will 

BEAD get consensus? M – 3

OECD Method (SOP 

MB-25) Carrier treatment Product application

This needs further discussion to see whether the use 

of a positive displacement pipette for product 

application is equivalent. M - 3

SOP MB-09-04 Carrier Wiping Number of wipes (passes)

EPA is currently using 6 passes; other labs are 

using 4 due to CA “requirements”. The number 

of passes must be standardized. Action item – 

follow-up is necessary to seek consensus on the 

number of passes across all labs. 

No issue with the use of 6 passes. Who does BEAD 

refer to as all labs? M – 2

SOP MB-09-04 

(Modified AOAC GSPT 

for Towelettes) Location of the test

EPA conducts the method inside the BSC while 

industry conducts the assay on the open bench. 

No action required; however, industry indicates 

that ease of towelette manipulation is greater on 

the open bench.

It may be easier to perform some tests on the lab 

bench. The canister is shook prior to removing 3-4 

wipes that are discarded before the one for testing is 

removed. 

http://www.cdc.gov/biosafety/publications/bmbl5/BM

BL.pdfC L

SOP MB-09-04 Carrier Inoculation

Use of positive displacement pipettes 

for carrier inoculation

EPA uses calibrated positive displacement 

pipettes and recommends their use for spotinoculation purposes whenever possible. 

Industry currently does not use them

Industry could discuss the use of positive 

displacement pipettes. There may be some labs 

that use them for inoculation. Discussion can be 

added to the method. L

SOP MB-09-04 Carrier Inoculation Spreading the inoculum

EPA uses flamed 4 mm loops, others use bent 

wire needles. No action required. Achieving the carrier count is what is important. L

SOP MB-09-04 Carrier Wiping Discarding towelettes

The number of towelettes discarded prior to 

beginning the official test is not described in the 

SOP. Action item – indicate the removal of 3-4 

towelettes prior to beginning study in the SOP

The removal of towelettes prior to testing does not 

take into account small count sizes or single packs. 

These smaller size packaging options need to be 

discussed. L

SOP MB-09-04 Carrier Wiping Towelette folding – 5 folds

Slight variations noted; however, as long as 

fresh surface is exposed per slide there is no 

issue. No action required

The folding technique was familiar to a few – may 

be some variation but the important part is to have a 

fresh area for each slide and wipe 10 

slides/towelette L

SOP MB-09-04 Testing Testing

Industry performs the method in two 30-carrier 

sets, while EPA conducts a single 60 carrier test. 

No action required; however, advantages and 

disadvantages should be explored for each 

approach. 

One difference is that the carriers do not sit at room 

temperature as long for a 30 slide test. Also BEAD 

does tests in sets of 20. Sixty carriers are 

inoculated and dried. How long do the carriers set 

before the last test is run? L

SOP MB-09-04 Overall

EPA recommends the preparation of an AOAC 

method specifically designed for towelettes 

using the AOAC spray test method (AOAC 

method 961.02) as the foundation with 

appropriate consensus-based modifications. 

Action item – is there agreement to pursue this 

activity?

This is the basis for the towelette method for 

industry. L

Draft Quantitative Petri 

Plate Method

Carrier inoculation and 

inoculum recovery Carrier inoculation

Use of template showing inoculation sites and 

positive displacement pipette is encouraged. No 

action required. This is a good practice. L

Draft Quantitative Petri 

Plate Method

Carrier inoculation and 

inoculum recovery Dislodging the inoculum

EPA demonstrated the process using a small 

scraper; however, the use of a larger scraper 

was encouraged by the group. Action item - 

EPA will explore the use of other devices. 

Larger scraper option is Thermo Scientific/Nunc 

#179693 L

Draft Quantitative Petri 

Plate Method

Carrier inoculation and 

inoculum recovery 40 mL suspension

The group recommends rinsing the tube 

containing the 40 mL suspension with saline or 

PBS and processing the rinsate. Action item – 

revise the draft method to include a rinsing step. This is a good recommendation. L

Draft Quantitative Petri 

Plate Method

Carrier inoculation and 

inoculum recovery Control counts 

Ensure use of the neutralizer as the solution 

applied to control plates; no action required, the 

draft procedure currently states use of the 

neutralizer. L

Use-dilution method 

(SOP MB-05)

Culture harvest and 

carrier inoculation

Harvest of Pseudomonas using 20 mL 

test cultures

Careful manipulation of culture is required to 

avoid disruption of the pellicle. This can be 

achieved through aspiration of the broth and/or 

removal of the pellicle itself. Easier 

manipulation in 20 mL tubes. No action items. 

CSPA members thought that the growth looked light 

and the pellicle didn’t look as thick as they see in 

their labs. L

Use-dilution method 

(SOP MB-05)

Bioscreening and 

efficacy testing Bioscreening

Visual physical screening performed in advance 

of bioscreening. Failures in the UDM are always 

separated and rescreened prior to use. Control 

carrier counts are not considered failed carriers. 

No action items. 

This follows industry practice. There wasn’t any 

discussion. L

Use-dilution method 

(SOP MB-05)

Bioscreening and 

efficacy testing Efficacy test (disinfectant tubes)

Use of Bellco 25 x 100mm tubes encouraged by 

EPA to reduce potential for contamination of 

inside of disinfectant tubes – catalog part 

number provided to participants. Carrier 

repeated (new carrier) if analyst inadvertently 

touches inside of the tube. No action items.

Belco 2010-25100 25x125mm - $45/144 tubes 

CSPA would like to confirm this information from 

BEAD.

L

OECD Method (SOP 

MB-25) Screening

Perform screening (reduce carrier numbers) in 

advance to devise dilution series. No action 

items. This is screening of the product L

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. Action 

item – further discussion required.

The Test Guidelines have been changed to have all 

organisms prepared the same way. L

OECD Method (SOP 

MB-25) Collaborative study

EPA expressed interest in conducting 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 as the collaborative 

study is developed. 

CSPA companies indicated that some would be 

interested in taking part in the collaborative. L