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Send to: Dr. Preetha Biswas
Neogen Corporation
620 Lancaster Place
Lansing, MI 48912
Result: COMPLETE Report Date: June 12, 2017
Customer Name: Neogen Corporation
Location of Testing: NSF Ann Arbor
Description: Listeria Right Now-Validation Study
Test Type: Test Only
Job Number: J-00253638
Project Number: 10058523
NSF Corporate: C0187278
Project Manager: K. Martin
Executive Summary: Neogen Corporation requested NSF International to perform a validation study to evaluate the
performance of the Listeria Right Now Assay (LRN) for detection of Listeria spp. In environmental swabs without
enrichment.
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: ____________________________________________
Jesse Miller – Director, Applied Research Center
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Experimental Summary:
Target microorganisms:
Listeria monocytogenes ATCC 19115
Method Development:
To determine the most suitable method for culture preparation a comparison of bacterial titers between the uses of a
McFarland standard and historical expected titer.
1. A portion of a 16-18 h overnight culture of L. monocytogenes was washed by centrifugation and re-suspended in
sterile 0.85% saline solution to the equivalent of a 4.0 McFarland standard.
2. Spread plate densities were then taken of the 4.0 McFarland Suspension and compared to the overnight culture
alone.
3. NSF determined that the concentration of the L. monocytogenes test strain is 5 times lower than the turbidity
standard that the provided product literature suggests. The lab adjusted the spike densities accordingly to more
accurately meet target spike densities specified by the client.
Environmental Surface Study:
Cleaning:
Stainless steel surfaces were cleaned according to the following procedure before testing:
1. Clean the stainless steel surface with alkaline detergent (or 1N NaOH), by soaking Kimwipes with detergent and
scrubbing (wipe forcefully) the surface and then rinsing with DI water.
2. Spray 10% fresh bleach (prepared within 24 h of use) on the surface, wait for 10 min, then scrub and Kimwipe.
3. Rinse the surface with deionized water and allow to air dry.
4. Spray 70% isopropyl alcohol on the surface, and wait for 5minutes.
5. Rinse with sterile deionized water and allow to air dry.
Culture Preparation:
All culture preparation was conducted using the following method:
1. Observe the table below for the inoculum size and number of replicates.
*Background organisms: A cocktail of Pseudomonas aeruginosa, Bacillus subtilis, and Enterococcus faecium at 1x104
CFU/mL
Listeria monocytogenes (CFU/mL)
Organisms Negative (Positive)
1x104
(Level 1)
8
(Level 2)
24
(Level 3)
60
Total
(Excluding
Negative)
L. monocytogenes +
Background* 5 5 15 15 15 50
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2. L. monocytogenes Prep
a. Centrifuge a 16-18 h overnight culture of Lm 19115 at 3500Xg for 15 minutes and suspend in 1 mL
0.85% saline solution
b. Prepare a 4.0 McFarland Standard in 0.85% saline solution from the centrifuged stock culture.
i. Theoretical concentration: 1.2x109 CFU/mL.
c. Dilute 4.0 McFarland solution to 1x105, 600, 240 and 80 CFU/mL in BPW.
i. Create the 1x105 CFU/mL stock with a final volume of 10mL.
ii. Pipette 1mL of the 4.0 McFarland in 9mL BPW, to make 10-1.
iii. Pipette 1mL of the 10-1 in 9mL BPW, to make 10-2.
iv. Pipet 417µL of 10-2 in 9.6mL BPW, to make 1x105 CFU/mL.
d. Create the 600 CFU/mL stock with a final volume of 40 mL.
i. Pipet 0.1mL of the 4.0 McFarland in 9.9mL BPW, to make 10-2.
ii. Pipette 0.1mL of the 10-2 in 9.9mL BPW, to make 10-4.
iii. Pipette 1mL of 10-4 in 39mL BPW, to make 600 CFU/mL.
e. Create the 240 CFU/mL stock by combining.
i. Pipette 4mL of 600 CFU/mL in 6mL BPW, to make 240 CFU/mL.
f. Create the 80 CFU/mL stock by combining the following:
i. Pipette 1.333 mL of 600 CFU/mL in 8.7mL BPW, to make 80 CFU/mL.
g. Add 1 mL of each diluted concentration of L. mono (1x105, 600, 240 and 80 CFU/mL) respectively, to 4
tubes containing 9 mL BPW.
h. The resulting volume will be 10 mL with the following final concentrations:
i. L. mono: 1x104, 8, 24 and 60 CFU/mL in BPW
3. L. monocytogenes + Background Prep
a. Centrifuge a 16-18 h overnight cultures of Pa 10145, Bs 9372, and Ef 19434 at 3500Xg for 15 minutes
and suspend in 1 mL 0.85% saline solution.
b. Prepare a 1.0 McFarland Standard of each organism in 0.85% saline solution from the centrifuged stock
culture.
i. Theoretical concentration: 3.0x108 CFU/mL.
c. Dilute each 1.0 McFarland standard solution 1:1000 in BPW.
i. Theoretical concentration 3.0x105 CFU/mL.
d. Pool all organisms into a single tube.
e. To 4 tubes of 8 mL BPW, add 1 mL of the organism pool. The resulting volume will be 9 mL.
f. Add 1 mL of each diluted concentration of L. mono (1x105, 600, 240 and 80 CFU/mL) respectively, to the
4 tubes containing the 9 mL diluted background organism cocktail.
g. The resulting volume will be 10 mL with the following final concentrations:
i. L. mono: 1x104, 60, 24 and 8 CFU/mL in BPW.
ii. Background: 1.0x104 CFU/mL in BPW.
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4. Inoculum count: Once the spike culture has been prepared, perform spread plating of the spike. Observe the table
below for dilutions and replicates to plate. Perform the plating as follows:
a. Right after preparing the inoculum (pre-spike), and
b. After spiking all the surfaces (post-spike)
5. An aliquot of 0.25mL of the inoculum is applied on an area of 4” x 4” ± 0.5” per surface and spread using a sterile
spreader.
6. Spike 2 square surfaces and spread using a sterile spreader. Wait for 1 minute. Spike the next 2 square surfaces.
a. If there are no squares with 50% dried inoculum, repeat this spiking pattern for the next pair of squares.
Processing:
1. Spiking, quality control and processing was carried out in accordance with “Appendix B: Listeria Right Now
Assay- Validation Study Plan for Independent Laboratory”.
Additional testing parameters:
All test swabs were stored at 4o C for a minimum of 30 minutes before testing with the ANSR assay.
Inoculum
count Solutions Organisms Dilution
Replicate
plates/dilution
Pre-spike
McFarland Standard
L. monocytogenes 10-6, 10-7 2
Background org 1 10-6, 10-7 2
Background org 2 10-6, 10-7 2
Background org 3 10-6, 10-7 2
Pooled Background 10-3, 10-4 2
60 CFU/mL Inoculum L. monocytogenes 0.25 mL 3
60 CFU/mL Inoculum +
1.0x104 CFU/mL background
cocktail
Total 10-2, 10-3 2
Post-spike
60 CFU/mL Inoculum L. monocytogenes 0.25 mL 3
60 CFU/mL Inoculum +
1.0x104 CFU/mL background
cocktail
Total 10-2, 10-3 2
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Summary/Conclusion
The purpose of this study was to evaluate the performance of the Listeria Right Now (LRN) assay for the detection of
Listeria spp. in environmental swabs without a prior enrichment process.
The surfaces of 4” x 4” squares of food-grade stainless steel were inoculated with different levels of Listeria monocytogenes
and a consortium of competing organisms, including Pseudomonas aeruginosa, Bacillus subtilis, and Enterococcus
faecium. After allowing the inoculum to partially dry (50%), surface samples were collected using semi-paired swabs. One
swab was tested by the Listeria Right Now assay and the other swab was enriched by the culture method. The swab for the
culture method was enriched overnight at 37°C in growth medium and an aliquot plated on to agar plates for detection on
the following day.
In the Listeria Right Now test, the entire collected contents of the swab were subjected to sample processing and testing on
the same day. After expression of the swab in the lysis buffer, one-half of the volume (0.5 mL) was taken for the lysis
incubation steps. Next, a portion of the lysed sample was transferred to a strip tube containing lyophilized reaction reagents.
The tubes were sealed and incubated at 56°C on the Neogen reader. Results generated by the reader were displayed in the
LRN software.
No false negatives, false positives or invalids were observed during this study. The evaluation determined that under the
conditions employed in this study, the enrichment-free Listeria Right Now method is as sensitive as the enrichment-based
culture reference method for detection of L. monocytogenes on a stainless steel surface.
Test Notes:
Additional inoculum counts were taken during surface testing to adequately examine bacterial titers during and after
spiking test surfaces.
MOX agar was outside of the pH range of 7.0 +/- 0.2. Quality control was performed on the agar batch and it
passed.
The McFarland suspensions were first prepared in 0.85% sterile saline solution before being diluted in BPW. This
was due to coloration present in the BPW that would have affected the preparation of the McFarland suspension.
References:
Neogen ANSR User Manual
Scope of Work Revisions:
Scope of work authorized on March 7, 2017.
Version 10058523 1 04052017 authorized on April 5, 2017 contained the following revisions:
o Updated costs associated to revised scope of work
o Updated timeline associated to revised scope of work
o Updated Appendix B protocol per edits made by Bryan Schindler on 4/4/2017
o Updated footer to include page numbering new (project) version number 10058523 1
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Results
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Table 1: Method development results to determine the most suitable method for culture preparation by comparing
bacterial titers between the uses of a McFarland standard and historical expected titer. TNTC= too numerous to count.
Table 2: Environmental surface study inoculum counts.
*Possible lab accident occurred during the enumeration of “L. monocytogenes (target 60 CFU/mL) + Background”.
**The concentration of the Background Cocktail in the inoculum suspension was 5.10E+03 CFU/mL; 10-fold less than
the Background Cocktail 10X stock (5.10E+04 CFU/mL).
Inoculum Prep type Theoretical CFU/Plate Actual CFU/Plate
McFarland Standard TNTC
Historical Density 201
Inoculum Prep type Theoretical CFU/Plate Actual CFU/Plate
McFarland Standard 3
Historical Density 46
Inoculum Prep type Theoretical CFU/Plate Actual CFU/Plate
McFarland Standard 1 15
McFarland Standard 2 13
Historical Density 8
Round One
15
15
15
Round Three
Round Two
Inoculum Count CFU/mL
L. monocytogenes (McFarland std) 4.60E+08
P. aeruginosa (McFarland std) 1.20E+09
B. subtilis (McFarland std) 2.50E+07
E. faecium (McFarland std) 3.14E+09
Background Cocktail 10X stock 5.10E+04
Inoculum Count (pre-spike) CFU/mL
L. monocytogenes (target 60 CFU/mL) 34
L. monocytogenes (target 60 CFU/mL) + Background 1.00E+03
Inoculum Count (post-spike) CFU/mL
L. monocytogenes (target 60 CFU/mL) 56
L. monocytogenes (target 60 CFU/mL) + Background 7.10E+03
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Table 3: Environmental surface study results for Listeria monocytogenes and background organisms.
Note: Inoculum spikes for levels 1, 2, and 3 contained 1.5E+03 CFU per coupon (theoretical 7.6E+02 CFU per swab) of
background cocktail.
LRN If Retest MOX Conf LRN If Retest MOX Conf
1 Negative N/A Negative N/A 1 Positive N/A Positive Positive
2 Negative N/A Negative N/A 2 Positive N/A Positive Positive
3 Negative N/A Negative N/A 3 Positive N/A Positive Positive
4 Negative N/A Negative N/A 4 Positive N/A Positive Positive
5 Negative N/A Negative N/A 5 Positive N/A Positive Positive
1 Positive N/A Negative N/A 1 Positive N/A Positive Positive
2 Positive N/A Positive Positive 2 Positive N/A Positive Positive
3 Positive N/A Positive Positive 3 Positive N/A Positive Positive
4 Positive N/A Positive Positive 4 Positive N/A Positive Positive
5 Positive N/A Positive Positive 5 Positive N/A Positive Positive
6 Positive N/A Positive Positive 6 Positive N/A Positive Positive
7 Positive N/A Negative N/A 7 Positive N/A Positive Positive
8 Positive N/A Negative N/A 8 Positive N/A Positive Positive
9 Positive N/A Positive Positive 9 Positive N/A Positive Positive
10 Positive N/A Negative N/A 10 Positive N/A Positive Positive
11 Negative N/A Positive Positive 11 Positive N/A Positive Positive
12 Positive N/A Positive Positive 12 Positive N/A Positive Positive
13 Positive N/A Negative N/A 13 Positive N/A Positive Positive
14 Positive N/A Positive Positive 14 Positive N/A Positive Positive
15 Positive N/A Negative N/A 15 Positive N/A Positive Positive
1 Positive N/A Positive Positive
2 Positive N/A Positive Positive
3 Positive N/A Positive Positive
4 Positive N/A Positive Positive
5 Positive N/A Positive Positive
6 Positive N/A Positive Positive
7 Positive N/A Positive Positive
8 Positive N/A Positive Positive
9 Positive N/A Positive Positive
10 Positive N/A Positive Positive
11 Positive N/A Positive Positive
12 Positive N/A Positive Positive
13 Positive N/A Positive Positive
14 Positive N/A Positive Positive
15 Positive N/A Positive Positive
NegativePositive: 4.8E+4 CFU/coupon
(Theoretical 2.4E+4 CFU/swab)
Level 1: 6 CFU/coupon
(Theoretical 3 CFU/swab)
Level 2: 18 CFU/coupon
(Theoretical 9 CFU/swab)
Level 3: 45 CFU/coupon
(Theoretical 22.5 CFU/swab)
Listeria monocytogenes +Background Organisms
Swab 1 Swab 2 Swab 1 Swab 2
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Table 4: Environmental surface study results for Listeria monocytogenes and background organisms on stainless steel.
Level
Theoretical
Inoculum
(CFU/swab)
Sample
Number
LRN
Positive
% LRN
Positive
Culture
Positive
% Culture
Positive
Negative 0 5 0 0% 0 0%
Positive 2.4E+4 5 5 100% 5 100%
L1 3 15 14 93% 9 60%
L2 9 15 15 100% 15 100%
L3 22.5 15 15 100% 15 100%
Note: Table 4 presents the results for the environmental surface study using a challenge inoculum of L. monocytogenes
plus a consortium of competing organisms. Three different inoculation levels were evaluated on the stainless steel
carriers: Level 1 = 3 CFU, Level 2 = 9 CFU and Level 3 = 22.5 CFU (theoretical CFU/swab). At Level 1, the detection
rates for LRN and the reference enrichment-based culture method were 93% and 60%, respectively. At Levels 2 and 3,
the detection rates for LRN and the reference enrichment-based culture method were 100%. No false negatives, false
positives or invalids were observed during this study. The data illustrates that under the conditions employed in this study
Listeria Right Now is as sensitive as the enrichment-based culture reference method for detection of L. monocytogenes on
a stainless steel surface.
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Appendix A Listeria Right Now Assay-Validation Study Plan for Independent Laboratory
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Appendix B NSF Terms and Conditions
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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
EPORT