NSF Final Report

NSF International Applied Research Center

789 N. Dixboro Rd. Ann Arbor, MI 48015, USA 1-800.NSF.MARK | +1-734.769.8010 | www.nsf.org

FI20150713100356 J-00175137 Page 1 of 21

Written NSF approval is required for reproduction of this report. Only authorized reports in their entirety may be distributed. This report does not represent authorization to use the NSF Mark. NSF Certification may be confirmed at www.nsf.org. The results of this report relate only to those items

tested.

TEST REPORT 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 Neogens 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. Neogens 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.

Report Authorization:____________________________________________ Robert Donofrio Director, Applied Research Center



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tested.

TEST REPORT 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 4x4 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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TEST REPORT Section 3: ATP was recovered from a concentrated spot randomly located on 4x4 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 systems 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 4x4 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(1partorangejuiceto999partssterilewater), 1:5000(1partorangejuiceto4999partssterilewater)and 1:10000(1partorangejuiceto9999partssterilewater).

*Real world approach is defined wherein each 4X4 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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TEST REPORT 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 4x4

stainless steel surface. Section 3 evaluated the recovery of a concentrated spot of ATP randomly located on a

4x4 stainless steel surface and Section 4 involved assessing the recovery of orange juice (commodity testing)

deposited over a 4x4 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 L 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 4x4 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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tested.

TEST REPORT 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 4x4 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 4x4 surface and air dried for 1 hour at room temperature (18-

25C) to measure the amount of ATP that could be recovered utilizing each monitoring system. This was accomplished by pipetting 20 L 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 4x4 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 manufacturers

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 20L

of the 5.0 nM ATP solution (100 femtomoles) was pipetted at a random spot on a 4x4 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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tested.

TEST REPORT Surfaces for each dilution level were prepared by dispensing 50 L of a given dilution level across the surface of

a 4x4 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 L 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 L onto the samplers. Results are reported in Table 4.



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tested.

TEST REPORT 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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tested.

TEST REPORT 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 2xs 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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tested.

TEST REPORT 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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tested.

TEST REPORT

Appendix A

Result Tables



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tested.

TEST REPORT 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)

Reader Neogen AccuPoint 3.04 3M NG

Hygiena EnSure

Charm NovaLUM

BioControl MVP

Sampler

AccuPoint Advanced

CleanTrace

UltraSnap

PocketSwab Plus

Lightning

Mean RLU

593.32

871.56

206.64

29,809.04

594.12



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tested.

TEST REPORT Table 2. Recovery of ATP standards from a homogenously contaminated stainless steel surface. A 100 femtomole ATP standard solution was homogenously deposited over a 4x4 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.

Reader Neogen 3M Hygiena Charm BioControl

AccuPoint 3.04 NG EnSure NovaLUM MVP

Samplers AccuPoint Advanced

UltraSnap PocketSwab

Plus Lightning CleanTrace

Mean RLU Recovered

from Surface

Average 165.2 62.8 31.1 8,618.10 123.7 Std Dev 34.87 20.08 18.62 5,236.99 47.34

%CV 21.11% 31.98% 59.86% 60.77% 38.27% % ATP

Recovery from

Surface

Average 27.84% 7.21% 15.05% 28.91% 20.82%



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TEST REPORT

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

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

0

5

10

15

20

25

30

35

Neogen 3M Hygenia Charm Biocontrol

%Recovery

%Recovery

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%


%CoefficientVariance

%CoefficientVariance



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tested.

TEST REPORT 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 4x4 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.

Reader Neogen 3M Hygiena Charm BioControl

AccuPoint 3.04 NG EnSure NovaLUM MVP

Samplers AccuPoint Advanced

UltraSnap PocketSwab

Plus Lightning CleanTrace

Mean RLU Recovered

from Surface

Average 240.3 71.3 20 3,435.40 106.5 Std Dev 97.51 45.34 13.25 2,900.68 88.79

%CV 40.58% 63.58% 66.25% 84.43% 83.37% % ATP

Recovery from

Surface

Average 40.50% 8.18% 9.68% 11.52% 17.93%



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tested.

TEST REPORT

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

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.

0

5

10

15

20

25

30

35

40

45


%Recovery

%Recovery

0

5

10

15

20

25

30

35

40

45


%Recovery

%Recovery



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tested.

TEST REPORT Table 4a. RLU values observed when the dilutions were amended directly to the ATP monitoring systems 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

Samplers

Orange Juice

Dilution

Neogen AP

Advanced 3M

CleanTraceHygiena

UltraSnap

Charm PocketSwab

Plus BioControl 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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TEST REPORT Table 4b. RLU values observed from sampling 4x4 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

Samplers Orange Juice

Dilution Neogen AP Advanced

3M CleanTrace

Hygiena UltraSnap

Charm PocketSwab

Plus BioControl Lightning

Average

1:1,000

553.3 71.4 65.7 14,468.30 271

% ATP Recovery 31.03% 1.97% 10.27% 9.93% 13.08%

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

Average

1:5,000

119.8 48.2 27.9 2,115.40 148.1

% ATP Recovery 28.63% 5.79% 16.85% 6.13% 25.43%

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

Average

1:10,000

14 26.6 0 14.4 10.5

% ATP Recovery 15.44% 12.23% 0.00% 0.23% 7.51%

%CV 146.7 111.52 NA 316.2 31.2



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tested.

TEST REPORT 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



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TEST REPORT

Appendix B

Sanitation Sampler Assay Protocol



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tested.

TEST REPORT 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 (18-23C) according to the manufacturers 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 M should be verified by UV (ATP 259 = 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 -20C 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 -20C 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 4x4 stainless steel surface. Section 3 is recovery of a concentrated spot of ATP randomly located on a 4x4 stainless steel surface and section 4 is recovery of orange juice (commodity testing) from a 4x4 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 L 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, 4x4 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 4x4 plate should be checked for cleanliness using an AccuPoint sampler to ensure the reading is at background (below 25 RLU).



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tested.

TEST REPORT 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 (18-25C). This is accomplished by pipetting 20 L 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 4x4 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 4x4 stainless steel surface, 20L 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 L of a given dilution level across the surface of a 4x4 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 L 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 L onto the samplers.

2015-07-13T10:21:51-0400Dr. Robert Donofrio /jv

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