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TEST REPORT

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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



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


McFarland Standard 3



McFarland Standard 1 15

McFarland Standard 2 13


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





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









11 Negative N/A Positive Positive 11 Positive N/A Positive Positive





1 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)


(Theoretical 9 CFU/swab)


(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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