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Send to: Neogen Corporation
620 Lesher Place
Lansing, Michigan 48912

Result: COMPLETE Report Date: 13-July-2015

Customer Name: Neogen Corporation

Location of Testing: NSF Ann Arbor
Description: Sanitation Sampler Assay Protocol - Real World Study
Test Type: Test Only
Job Number: J-00175137
Project Number: 10009584
NSF Corporate: C0245040
Project Manager: J. Vantine

Executive Summary:
Neogen Corporation contracted the Applied Research Center at NSF International to perform comparison
performance testing of Neogen’s ATP system, AccuPoint Advanced, against four other commercially available
systems. The recovery efficiencies and consistency of each system were evaluated against an ATP standard
solution and orange juice at different dilutions inoculated onto stainless steel carriers. This study utilized several
methods to determine the effectiveness of each system. Both a directly pipetted ATP standard solution and
commodity onto swab surfaces and surface swabbing of stainless steel coupons were employed with the test
systems.
Neogen’s AccuPoint Advanced ATP system consistently yielded the highest percent recoveries and the most
consistent readings of the target analytes, when compared to the other four test systems.
ATP devices are utilized to detect the presence of bacteria and organic/food residue on surfaces. ATP has been
incorporated as a key monitoring parameter for the food, beverage and healthcare industries. It is essential that
these devices provide precise and consistent readings so that the hygiene practices of these industries can be
accurately evaluated.

Thank you for having your product tested by NSF International.

Please contact your Project Manager if you have any questions or concerns pertaining to this report.
Digitally signed by Dr. Robert Donofrio /jv
DN: cn=Dr. Robert Donofrio /jv, o=NSF
International, ou=Director - Applied Research
Center, [email protected], c=US
Report Authorization:____________________________________________
Date: 2015.07.13 10:21:51 -04'00'

Robert Donofrio – Director, Applied Research Center

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Scope of Test Report

Adenosine triphosphate (ATP) is a compound found in every living cell and can be used as an indicator to
determine if a surface was properly sanitized. ATP hygiene monitoring systems have been used in the food
production industry for over twenty years. The systems are used in facilities to measure the cleaning
effectiveness, removal/reduction of ATP, on food contact surfaces. Our study attempts to mirror typical field
usage by looking at the recovery of each system of ATP standards and commodity from a common surface
(stainless steel.)
Multiple manufactures produce monitoring systems to detect ATP. The following systems were selected to be
tested in this study.

 Neogen AccuPoint Advanced ATP Surface Samplers (Lot 216036)

 3M Clean-Trace Surface ATP (Lot 1222C)
 Hygiena UltraSnap (Lot 02515)
 Charm PocketSwab Plus (Lot 4031315A-01)
 Biocontrol LIGHTNING MVP ICON ATP Surface Sampling Device (Lot 042915-01)

Neogen Corporation provided its system and the reference standard ATP, all other devices were purchased
independently through normal commercial channels by NSF International.

Each system was tested using 4 differing approaches. Details provided in Methodology.

Section 1: ATP standard solutions were pipetted directly onto sample swabs. Data obtained here was used as a
reference for the calculation of ATP recovery in sections 2 and 3. (Data recorded under NSF J-00170817)

Section 2: ATP standards were deposited over a 4”x4” stainless steel surfaces and the above referenced
monitoring systems were used to sample the entire surface under a real world approach*.

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Section 3: ATP was recovered from a concentrated spot randomly located on 4”x4” stainless steel surface
utilizing all 5 test systems detailed above with a real world swabbing approach. In the real world, residue is not
homogeneously distributed across a surface after cleaning. The spot test attempted to recreate this phenomenon by
randomly placing a small, invisible dot of ATP on the stainless steel surfaces to determine the extent and
consistency of each system’s ability to locate and accurately identify its presence. 10 replicates of each test were
completed on a single test day for each reader.

Section 4: Orange juice was used as a standard commodity to measure the detection ability of each of the test
units. Orange juice was deposited over a 4”x4” stainless steel surface that was sampled utilizing all 5 test systems
detailed above with a real world swabbing approach.. 10 replicates of each test were completed on a single test
day for each reader.

The following series of dilutions were tested:

 1:1000 (1 part orange juice to 999 parts sterile water),  
 1:5000 (1 part orange juice to 4999 parts sterile water) and  
 1:10000 (1 part orange juice to 9999 parts sterile water).   

*Real world approach is defined wherein each 4”X4” surface was sampled in a cross-hatch pattern with five
seconds in each direction. This measurement was adapted to replicate real-world operation of these systems that
most often occurs between tightly scheduled production runs where sampling speed is important.

Methodology
These following methods were derived from document “Sanitation Sampler Assay Protocol,” provided by Brent
Steiner and Ron Sarver of Neogen Biochemistry Laboratory on December 30, 2014. (See Addendum B for
complete text)

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Recovery Evaluation Protocols
Evaluations of the sanitation systems were conducted in 4 sections. Section 1 involved the addition of ATP
standard solutions directly to sample swabs. Section 2 evaluated the recovery of ATP deposited over a 4”x4”
stainless steel surface. Section 3 evaluated the recovery of a concentrated spot of ATP randomly located on a
4”x4” stainless steel surface and Section 4 involved assessing the recovery of orange juice (commodity testing)
deposited over a 4”x4” stainless steel surface. Each section is described in detail below. Adenosine 5’-
triphosphate disodium salt hydrate (ATP) reference standards were prepared and provided by Neogen Corporation
for use during these studies.

Section 1. The goal of this experimental section was to determine the Relative Light Unit (RLU) response for
each of the 5 test systems that corresponded to standard ATP solutions added directly to the sampling system. For
each sanitation system, 20 uL of each ATP standard (0, 12.5, 25.0, and 100 femtomoles of ATP) was pipetted
directly onto the sample swab or pad of the sanitation system. Immediately following addition of the ATP
standard to the sample pad or swab the instructions for the system were followed and the sampler was read on the
appropriate luminescence reader. Each ATP concentration including a blank (sterile water) was tested 25 times
using 25 different samplers. The ATP solutions were labeled by nanomolar concentration and 20 µL of the 5.00,
1.25 and 0.625 nM solutions of ATP or sterile water result in the following femtomoles of ATP on the sample pad
or swab, 100, 25.0, 12.5 and 0 femtomoles, respectively. Results for the 100 femtomole solution are reported in
Table 1. The calculated mean RLU response for the 100 femtomole solution was used as a reference for
calculating ATP recovery for the experiments performed in Sections 2 and 3.

Section 2. The goal of this experimental section was to determine the efficiency of the five test systems in
recovering an ATP standard that had been evenly spread across a stainless steel carrier. The surface recovery of
ATP or commodities was determined by using a 4”x4” stainless steel plate. The initial cleanliness of the stainless
steel plates was important to monitor and the testing was conducted in a laminar flow hood equipped with a UV
lamp. Prior to each round of testing the stainless steel plates were cleaned and sterilized using a UV lamp with a
twenty minute exposure. Between experiments the stainless steel plates were cleaned using 10% Contrad 70 in
water, then rinsed with sterile water, washed again with isopropanol and then air dried.

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Sterile water and isopropanol used for cleaning was dispensed from a spray bottle which was sterilized using a
UV lamp (20 minute exposure) and 10% Contrad 70 in water. Periodically a 4”x4” plate was checked for
cleanliness using an AccuPoint sampler to ensure the reading was at background levels (below 25 RLU).

100 femtomoles of ATP was spread over the 4”x4” surface and air dried for 1 hour at room temperature (18-
25C) to measure the amount of ATP that could be recovered utilizing each monitoring system. This was
accomplished by pipetting 20 µL of a 5.0 nM ATP solution onto the stainless steel surface. The tip of a pipette
was used to distribute the solution over the surface. Ten 4”x4” stainless steel squares were covered with 100
femtomoles of ATP for each sanitation system. After the ATP was deposited homogenously across and dried, the
surface was sampled using the sanitation system sampler in the manner recommended by the manufacturer’s
instructions. The amount of ATP was recovered was determined by comparing the mean response from the
surface recovery to the mean response obtained in Section1. Results are reported in Table 2.

Section 3. The goal of this experimental section was to determine the efficiency of the five test systems in
recovering an ATP standard that had been spot inoculated at a random location on a stainless steel carrier. A 20µL
of the 5.0 nM ATP solution (100 femtomoles) was pipetted at a random spot on a 4”x4” stainless steel surface to
determine the recovery capability of each monitoring system. The spot was allowed to dry for 1 hour and the plate
was sampled according to the manufacturers sampling instructions. This was repeated 10 times for each sanitation
monitoring system to determine the mean response, standard deviation and the coefficient of variation (CV) for
the recovered ATP from the surface. The percentage recovered from the surface was determined by comparing the
mean response from the surface spot recovery to the mean response obtained in Section 1. Results are reported in
Table 3.

Section 4. The goal of this experimental section was to determine the efficiency and detection limit of the five test
systems in recovering a standard commodity, orange juice, which had been evenly spread across a stainless steel
carrier. This experiment was designed to replicate a typical situation that would be encountered in the field. For
this evaluation, 10mL of orange juice was diluted 1:1000 (1 part orange juice to 999 parts sterile water), 1:5000 (1
part orange juice to 4999 parts sterile water), and 1:10000 (1 part orange juice to 9999 parts sterile water).

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Surfaces for each dilution level were prepared by dispensing 50 µL of a given dilution level across the surface of
a 4”x4” section of stainless steel plate and allowing the samples to dry for 1 hour before sampling the surface
according to the prescribed method for each brand of system. Ten surfaces were prepared and sampled at each
dilution for each brand of sanitation systems. The mean, standard deviation and coefficient of variation was
determined for each dilution and each brand of sanitation sampler.

To determine recovery, 50 µL of each orange juice dilution was pipetted directly onto the swab or sample pad and
the response measured using each brand of sanitation system. This was repeated ten times to determine the mean
response for directly pipetting the orange juice dilution onto the sampler. The percentage recovered from the
surface was determined by comparing the mean response from the surface recovery to the mean response obtained
from directly pipetting 50 µL onto the samplers. Results are reported in Table 4.

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Results and Discussion
A comparative evaluation of the ability of five commercial ATP monitoring systems to accurately report ATP
levels from stainless steel surfaces was performed. The studies examined the difference in recovery of ATP
standards when applied in a homogenous manner across the carrier as well as to a random spot contamination.
The study also assessed the ability of the five ATP monitoring systems to detect a standard commodity food,
orange juice, which was applied to carriers in varying concentrations. The results of the study are presented in
Appendix A.

During Section 1 of this study, the RLU (Relative Light Unit) outputs for the five test systems were observed
when ATP standards were directly introduced onto the swabs/sample pads. The mean RLU output was calculated
for 25 replicates and reported in Table 1. Table 1 contains the RLU observations for all five systems tested at the
100 femtomole ATP concentrations. The RLU data generated in Section 1 for was used as a reference for
calculating ATP recovery in Sections 2 and 3.

Section 2 of the study utilized stainless steel coupons prepared with the 100 femtomole of the reference ATP
standard as the sample. The surface was sampled using the monitoring systems’ operational instructions but
utilizing a real world approach to the exposure time of the swab contact on the sample surface. A standard run /
return pattern was used over the sample coupon on 2 axis/sides. Each side had the timed exposure of swab to
surface of 5 seconds making the entire exposure 10 seconds. This timeframe is relevant to compare the results of a
lab study to a real world, situational use of the monitoring system. The percent of ATP recovered was determined
by comparing the mean response from the surface recovery to the mean response of direct swab inoculation
observed in Section 1. Table 2 contains the results for the Section2 study. The two monitoring systems that had
the highest percent ATP recovery from the stainless steel surfaces were the Charm PocketSwab Plus (28.91%)
and the Neogen AccuPoint Advanced system (27.84%). The Neogen AccuPoint Advanced system also displayed
the lowest percent coefficient of variance (21.11%), indicating that it achieved the most consistent (least variable)
readings.

Section 3 involved assessing the ATP recovery efficiencies from stainless steel coupons with a random spot of 5.0
nM ATP solution of 100 femtomoles dried on it. The surfaces of 10 replicant coupons were sampled utilizing the

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real world situational sampling method utilizing each of the 5 monitoring systems to determine the mean response
of each unit.

The percentage recovery was calculated by comparing the mean response from the surface spot recovery to the
mean response of direct swab inoculation observed in Section 1. The results for Section 3 can be found in Table 3.
The Neogen AccuPoint Advanced had the highest percentage recovery of all 5 monitoring systems at 40.50%
recovery of the ATP solution from the unit surface. The Neogen AccuPoint Advanced system exhibited a percent
ATP recovery that was 2x’s greater than the next most efficient monitoring system. The Neogen Accupoint
system exhibited the greatest consistency in readings (with a CV of 21.11%), indicating that the system is very
precise. The next closest system was BioControl MVP system at 17.93% recovery.

In Section 4 the experimental protocol was designed to mimic real world contamination scenarios. This study
involved contaminating stainless steel surfaces with orange juice at 3 dilutions: 1:1,000, 1:5,000 and 1:10,000.
RLU reference values for each dilution were first generated by direct inoculation onto the ATP monitoring system
swabs. Recovery sampling using a real world approach, as previously described, was performed on
homogenously inoculated stainless steel surfaces. The percentage recovered from each surface was determined
by comparing the RLU of the surface reading with the RLUs observed from direct swab inoculation. Table 4
provides the results for the RLUs observed from direct inoculation (4a), RLUs from stainless steel recovery (4b)
and calculated percent ATP recovery (4c). Once again, the Neogen AccuPoint Advanced had the highest
observed percentage recovery of all 5 monitoring systems. For each of the orange juice dilutions evaluated, the
percent recovery of ATP by the Neogen AccuPoint Advanced was significantly higher than that of the other four
ATP monitoring systems evaluated. Once again, the Neogen Accupoint system proved to be the most consistent
of the devices evaluated (with a CV of 40.58%). The next closest system for recovery at 1:1000 and 1:5000
dilution factors was BioControl MVP. For the 1:10000 dilution factor the 2nd highest recovery was the 3M
CleanTrace.

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Scope of Work
 Scope of work authorized: April 30th, 2015
 No revisions were affected.

Disclosure of Deviations
On 5/08/2015 the stainless steel coupon cleaning was validated by 2 Neogen AP swab readings. The readings
were below the 25 RLU background thresholds (4 RLU, 11 RLU) and testing proceeded for the 10 replicate
samples of the AccupPoint 3.04, EnSure and MVP readers. However, each of the three readers had 1 or 2 outlier
samples indicating coupon contamination. Outlier is defined as a sample with an RLU value outside the range
observed when pipetting the orange juice directly on the swab.) After this day of testing, coupons were re-cleaned,
sampled for cleanliness (0 RLU, 0 RLU), and re-tested. Summary of results reported in Table 4 are the data
recovered on the retest date.

Contract Detail Clarification

NSF International and Neogen Corporation entered into a contractual relationship in February 2015. This
document, drafted by NSF International, is the Terms and Conditions for Testing, Audit and Evaluation Services.
Per section 11 of this agreement NSF authorizes Neogen to use select data charts/ graphs from this study in
publications. NSF requires that a draft of the usage be reviewed with the ARC program office / NSF Marketing
for final feedback and authorization to print.

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Appendix A

Result Tables

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Table 1. Mean RLU response of five ATP monitoring systems against an ATP standard of 100 femtomoles.
ATP standard solutions were pipetted directly onto sample swabs. Mean was taken of 25 replicates (n=25) and
reported. Data obtained was used as a reference for the calculation of ATP recovery in Sections 2 and 3, Tables 2
and 3 respectively. (Data recorded under NSF J-00170817)

Neogen 3M Hygiena Charm BioControl

Reader
AccuPoint 3.04 NG EnSure NovaLUM MVP

AccuPoint CleanTrace UltraSnap PocketSwab Plus Lightning

Sampler Advanced

Mean RLU 593.32 871.56 206.64 29,809.04 594.12

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Table 2. Recovery of ATP standards from a homogenously contaminated stainless steel surface. A 100
femtomole ATP standard solution was homogenously deposited over a 4”x4” stainless steel surface. The surface
was then sampled with the referenced monitoring systems utilizing a real world approach swabbing technique (5
seconds in one direction, and then swabbing 5 more seconds perpendicular to the first). The mean RLU was
calculated of 10 replicates (n=10) and reported. To calculate percent ATP recovery, the direct inoculation mean
RLU recovery values (Table 1) were used as a reference.

Neogen 3M Hygiena Charm BioControl

Reader AccuPoint
3.04 NG EnSure NovaLUM MVP
UltraSnap
AccuPoint PocketSwab
Samplers CleanTrace Lightning
Advanced Plus

Mean RLU Average 165.2 62.8 31.1 8,618.10 123.7

Recovered
from Std Dev 34.87 20.08 18.62 5,236.99 47.34
Surface %CV 21.11% 31.98% 59.86% 60.77% 38.27%
% ATP
Recovery
Average 27.84% 7.21% 15.05% 28.91% 20.82%
from
Surface

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% Recovery
35

30

25

20
% Recovery
15

10

0
Neogen 3M Hygenia Charm Biocontrol

Figure 1: provides a pictorial representation of Table 2: Recovery of ATP standards from a homogenously
contaminated stainless steel surface.

% Coefficient Variance
70.00%

60.00%

50.00%

40.00%
% Coefficient Variance
30.00%

20.00%

10.00%

0.00%
Neogen 3M Hygenia Charm Biocontrol

Figure 2: provides a pictorial representation of Table 2. Coefficient of Variance (%) was calculated and lowest %
indicates the most consistent (least variable) readings.

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Table 3. Recovery of an ATP standard from a single contamination spot on stainless steel surfaces. A 100
femtomole ATP standard solution was deposited randomly as a drop on a 4”x4” stainless steel surface. The
surface was then sampled with the referenced monitoring systems utilizing a real world swabbing approach
technique (5 seconds in one direction, and then swabbing 5 more seconds perpendicular to the first). The mean
RLU was calculated of 10 replicates (n=10) and reported. To calculate percent ATP recovery, the direct
inoculation mean RLU recovery values (Table 1) were used as a reference.

Neogen 3M Hygiena Charm BioControl

Reader AccuPoint
3.04 NG EnSure NovaLUM MVP
UltraSnap
AccuPoint PocketSwab
Samplers CleanTrace Lightning
Advanced Plus

Mean RLU Average 240.3 71.3 20 3,435.40 106.5

Recovered
from Std Dev 97.51 45.34 13.25 2,900.68 88.79
Surface %CV 40.58% 63.58% 66.25% 84.43% 83.37%
% ATP
Recovery
Average 40.50% 8.18% 9.68% 11.52% 17.93%
from
Surface

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% Recovery
45
40
35
30
25
20 % Recovery

15
10
5
0
Neogen 3M Hygenia Charm Biocontrol

Figure 3: provides a pictorial representation of Table 3: Recovery of an ATP standard from a single
contamination spot on stainless steel surfaces.

% Recovery
45
40
35
30
25
20 % Recovery

15
10
5
0
Neogen 3M Hygenia Charm Biocontrol

Figure 4: provides a pictorial representation of Table 3: Recovery of an ATP standard from a single
contamination spot on stainless steel surfaces. Coefficient of Variance (%) was calculated and lowest %
indicates the most consistent (least variable) readings. Table 4. Recovery of ATP from stainless steel surfaces
inoculated with varying concentrations of orange juice. Orange juice was selected as an ATP source since it is
a standard commodity product. Three dilutions of orange juice were utilized: 1:1,000, 1:5,000 and 1:10,000.

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Table 4a. RLU values observed when the dilutions were amended directly to the ATP monitoring system’s
sample pad/swab. These RLU values were used as a reference for the calculation of percent ATP recovery in
Table 4c. The average RLU reading from 10 replicates (n=10) is reported.

Mean RLU for Recovery of Orange Juice Pipetted onto the Sample Pad/ Swab
Orange Neogen Charm
Juice AP 3M Hygiena PocketSwab BioControl
Samplers Dilution Advanced CleanTrace UltraSnap Plus Lightning
Average 1:1000 1,783.4 3,629.1 639.6 145,735.9 2,071.9

Average 1:5000 418.5 832.6 165.6 34,517.6 582.4

Average 1:10000 90.7 217.5 34.0 6,394.1 139.9

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Table 4b. RLU values observed from sampling 4”x4” stainless steel surfaces amended with three dilutions of
orange juice. Percent recovery was calculated by dividing the mean RLU values below (homogenous stainless
steel coupon inoculation) by the mean RLU values in Table 4a (direct swab inoculation).
The dilutions were applied homogenously and the surfaces were sampled utilizing with a real world swabbing
approach (5 seconds in one direction, and then swabbing 5 more seconds perpendicular to the first). The average
RLU reading from 10 replicates (n=10) is reported.
Recovery of Orange Juice from 4"x4" Stainless Steel
Charm
Orange Juice Neogen AP 3M Hygiena PocketSwab BioControl
Samplers Dilution Advanced CleanTrace UltraSnap Plus Lightning

Average 553.3 71.4 65.7 14,468.30 271

% ATP
1:1,000 31.03% 1.97% 10.27% 9.93% 13.08%
Recovery

%CV 33.1%  74.3%  37.2%  47.6%  55.3% 

Average 119.8 48.2 27.9 2,115.40 148.1

% ATP
1:5,000 28.63% 5.79% 16.85% 6.13% 25.43%
Recovery

%CV 46.7%  32.0%  43.3%  36.5%  38.8% 

Average 14 26.6 0 14.4 10.5

% ATP
1:10,000 15.44% 12.23% 0.00% 0.23% 7.51%
Recovery

%CV 146.7 111.52 NA 316.2 31.2

FI20150713100356 J-00175137 Page 17 of 21

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Table 5: Stainless steel coupon cleaning readings were completed each test date by 2 Neogen AP Advanced
swabs of 2 representative coupons. All readings were at 0 RLU on days that data was reported from. Data
recorded on 5/8/2015 was dismissed due to outlier readings and repeated.

RLU
Replicate 1 Replicate 2
5/8/2015 4 11
5/11/2015 0 0
5/12/2015 0 0
5/13/2015 0 0
5/14/2015 0 0
5/15/2015 0 0
5/19/2015 0 0
5/20/2015 0 0
5/21/2015 0 0
5/22/2015 0 0

FI20150713100356 J-00175137 Page 18 of 21

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NSF International – Applied Research Center

789 N. Dixboro Rd. Ann Arbor, MI 48015, USA

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Appendix B

Sanitation Sampler Assay Protocol

FI20150713100356 J-00175137 Page 19 of 21

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Sanitation Sampler Assay Protocol
Neogen Biochemistry Laboratory
Brent Steiner and Ron Sarver
December 30, 2014

Materials
The ATP Sanitation readers used for this study should include Neogen AccuPoint v3.03, 3M Clean-Trace NG
ATP Illuminator, Charm Novalum, Hygiena Ensure and BioControl MVPICON. Readers should be turned on
and warmed up to room temperature (18-23C) according to the manufacturer’s instruction. Sanitation
monitoring samplers should include AccuPoint Advanced ATP Surface Samplers, 3M Clean-Trace Surface ATP,
Hygiena UltraSnap, Charm PocketSwab Plus and Biocontrol LIGHTNING MVP ICON ATP Surface Sampling
Device. Samplers also should be allowed to equilibrate to room temperature as recommended by the
manufacturer. ATP will be provided by Neogen. Solutions of ATP will be prepared by NSF for the study. If
additional ATP is required, analytical standard grade Adenosine 5’-triphosphate disodium salt hydrate (ATP) was
purchased from Sigma Chemical Co., part # FLAAS. ATP was dissolved in 50 mM Tricine buffer, pH 7.75, the
concentration of stock solution 50 µM should be verified by UV (ATP ε259 = 15.4 x 103 M-1 cm-1) and dilutions at
50.0 nM, 5.00 nM, 1.25 nM and 0.625 nM were prepared. Dilutions of ATP will be held on ice when not stored
in -20°C freezer. Pulp-free orange juice will be purchased and diluted in sterile water. Dilutions of orange juice
will be held on ice when not stored in -20°C freezer.

Section Protocols
Evaluations of the sanitation systems will be conducted in 4 sections. Section 1 is the addition of ATP standard
solutions directly to sample swabs. Section 2 is recovery of ATP deposited over a 4”x4” stainless steel surface.
Section 3 is recovery of a concentrated spot of ATP randomly located on a 4”x4” stainless steel surface and
section 4 is recovery of orange juice (commodity testing) from a 4”x4” stainless steel surface.

Section 1). For each sanitation system, 20 uL of each ATP standard (0, 12.5, 25.0, and 100 femtomoles of ATP)
is pipetted directly onto the sample swab or pad of the sanitation system. Immediately following addition of the
ATP standard to the sample pad or swab follow the instructions for the system and read the sampler on the
appropriate luminescence reader. Each ATP concentration including a blank (sterile water) is tested 25 times
using 25 different samplers. The ATP solutions are labeled by nanomolar concentration and 20 µL of the 5.00,
1.25 and 0.625 nM solutions of ATP or sterile water result in the following femtomoles of ATP on the sample pad
or swab, 100, 25.0, 12.5 and 0 femtomoles, respectively. For each sanitation monitoring system at each ATP
concentration, the mean response is calculated along with the standard deviation and coefficient of variation.

Section 2). For the determination of surface recovery of ATP or commodities, 4”x4” stainless steel plates should
be used. The cleanliness of the stainless steel plate is important and testing should be conducted in a laminar flow
hood equipped with a UV lamp. Prior to each round of testing the stainless steel plate should be cleaned and
sterilized using the UV lamp with twenty minutes exposure time. Between experiments the stainless steel plates
should be cleaned using 10% Contrad 70 in water, rinsed with sterile water, washed with isopropanol and air
dried. Sterile water and isopropanol used for cleaning will be dispensed from a spray bottle which has been
sterilized using a UV lamp (20 minutes exposure time) and 10% Contrad 70 in water. Periodically, a 4”x4” plate
should be checked for cleanliness using an AccuPoint sampler to ensure the reading is at background (below 25
RLU).

FI20150713100356 J-00175137 Page 20 of 21

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To determine the amount of ATP recovered from a stainless steel surface 100 femtomoles of ATP should be
spread over the 4x4” surface and air dried for 1 hour at room temperature (18-25C). This is accomplished by
pipetting 20 µL of a 5.0 nM ATP solution onto the stainless steel surface. The tip of the pipette can be used to
distribute the solution over the surface. Ten 4”x4” stainless steel squares should be covered with 100 femtomoles
of ATP for each sanitation system being evaluated. Care should be taken to not spread the solution with
something other than the pipette tip since other materials may remove the deposited ATP from the surface. After
the ATP is deposited and dried, the surface should be sampled using the sanitation system sampler in the manner
recommended by the manufacturer. The amount of ATP recovered is determined by comparing the mean
response from the surface recovery to the mean response obtained in Section 1 from directly pipetting 100
femtomoles of ATP onto the samplers.

Section 3). For recovery of a concentrated dot of ATP randomly spotted on a

4”x4” stainless steel surface, 20µL of the 5.0 nM ATP solution (100 femtomoles) is pipetted at a random spot on
each plate. The spot is then allowed to dry for 1 hour and the plate sampled according to the sampling method
prescribed by the manufacturer. This is repeated 10 times for each sanitation monitoring system to determine the
mean response, standard deviation and coefficient of variation (CV) for the recovered ATP from the surface. The
percentage recovered from the surface is determined by comparing the mean response from the surface spot
recovery to the mean response obtained in Section 1 from directly pipetting 100 femtomoles of ATP onto the
samplers..

Section 4). Commodity testing with orange juice is completed to determine recovery from a stainless steel surface
and determine the limit of detection in a more typical situation that would be encountered in the field. For this
evaluation, 10mL of orange juice is diluted 1:1000 (1 part orange juice to 999 parts sterile water), 1:5000 (1 part
orange juice to 4999 parts sterile water), and 1:10000 (1 part orange juice to 9999 parts sterile water). Surfaces
for each dilution level were prepared by dispensing 50 µL of a given dilution level across the surface of a 4”x4”
section of stainless steel plate and allowing the samples to dry for 1 hour before sampling the surface according to
the prescribed method for each brand of sampler. Care should be taken to not spread the solution with something
other than the pipette tip since other materials may remove the deposited orange juice from the surface. Ten
surfaces should be prepared and sampled at each dilution for each brand of sanitation systems. The mean,
standard deviation and coefficient of variation should be determined for each dilution and each brand of sanitation
sampler.
To determine recovery, 50 µL of each orange juice dilution should be pipetted directly onto the swab or sample
pad and the response measured using each brand of sanitation sampler. This is repeated ten times to determine
the mean response for directly pipetting the orange juice dilution onto the sampler. The percentage recovered
from the surface is determined by comparing the mean response from the surface recovery to the mean response
obtained from directly pipetting 50 µL onto the samplers.

FI20150713100356 J-00175137 Page 21 of 21

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