Performance Evaluation of HiCap TM Neutralizing Broth Monitoring/World...Performance Evaluation of...

of 22 This report is confidential and shall not be reproduced, except in full, without written authorization by

Cherney Microbiological Services, LTD.

Performance Evaluation of HiCapTM

Neutralizing Broth

March 1, 2013

Study Leader Teri Ehlinger/Microbiologist

Cherney Microbiological Services, LTD

Study Manager Joanne Ruebl/Project Manager


Study Sponsor World Bioproducts, LLC

PO Box 947 Bothell, WA 98041-0947

Tel: 877-260-6441; 425-242-4153

[email protected]

Cherney Microbiological Services, LTD Performance Evaluation of HiCapTM Neutralizing Broth



TABLE OF CONTENTS

1.0 Purpose ..................................................................................................................................... 3

2.0 Scope......................................................................................................................................... 3

3.0 Project Summary....................................................................................................................... 3

4.0 Materials and Methods............................................................................................................. 4

5.0 Results....................................................................................................................................... 5

6.0 Conclusions ............................................................................................................................... 6

7.0 Final Report Approval ............................................................................................................... 7

List of Tables

Table 1. Compatibility of HiCap with Commercial Pathogen Kits................................................. 6

Table 2. HiCap Neutralizing Broth and Butterfield’s Phosphate Buffer with 3M Petrifilm Plates 6

Appendix 1 – Raw Data Results of HiCap Neutralizing Broth with Commercial Pathogen Kits ..... 8

VIDAS Assay Results ............................................................................................................ 9

Salmonella Genus BAX Q7 Results.................................................................................... 10

Listeria Genus BAX Q7 Results.......................................................................................... 11

Salmonella Genus iQ-Check Results ................................................................................. 12

Listeria Genus iQ-Check Results........................................................................................ 13

Salmonella Genus ANSR Results ....................................................................................... 14

Listeria Genus ANSR Results ............................................................................................. 15

Appendix 2 – Pictures of 3M Petrifilm Plates after Incubation.................................................... 16

Picture Series 1. 3M Petrifilm Aerobic Count Plates Inoculated with HiCap Neutralizing

Broth and Bufferfield’s Phosphate Buffer ........................................................................ 17

Picture Series 2. 3M Petrifilm Enterobacteriaceae Count Plates Inoculated with HiCap

Neutralizing Broth and Butterfield’s Phosphate Buffer.................................................... 19

Picture Series 3. 3M Petrifilm Coliform Count Plates Inoculated with HiCap Neutralizing

Broth and Butterfield’s Phosphate Buffer ........................................................................ 21




1.0 PURPOSE

To evaluate the performance of HiCap Neutralizing Broth for an environmental monitoring

program.

2.0 SCOPE

This study determined the compatibility of HiCapTM Neutralizing Broth1 (or “HiCap”) with

selected commercial pathogen kits and with 3MTM PetrifilmTM 2 Plates.

3.0 PROJECT SUMMARY

This study assessed the compatibility EZ Reach Sponge Samplers (“EZ Reach Samplers”)

hydrated with HiCap Neutralizing Broth, with the following commercial pathogen kits:

• VIDASTM 3 (representing an automated immunoassay technology)

• RapidChekTM 4 (representing a lateral flow immunoassay technology),

• BAX System Q7TM 5 (representing a thermal cycling DNA amplification

technology),

• ANSRTM 6 (representing an isothermal DNA amplification)

• iQ-CheckTM 7

(representing a second thermal cycling DNA amplification

technology)

To perform this compatibility study, EZ Reach Samplers containing HiCap Neutralizing Broth

were inoculated with low levels (10-50 cells) of either Listeria spp. or Salmonella spp. The

inoculated EZ Reach Samplers were incubated for 72 hours at 4 +/- 1˚C and tested according to

the specific protocol for each pathogen kit. Additionally, HiCap Neutralizing Broth was

squeezed from the sponges of the EZ Reach Samplers and tested directly in the commercial test

kits (without including an enrichment step) to confirm that the solution itself does not cause

interference.

This study also evaluated the compatibility of the HiCap Neutralizing Broth with 3M Petrifilm

Plates. In the first part of the study, HiCap Neutralizing Broth was squeezed from the sponges

of the EZ Reach Samplers and the broth was inoculated with either Staphylococcus aureus,

Escherichia coli or Shigella flexneri. One ml aliquots of the inoculated HiCap Neutralizing Broth

was plated onto 3M Petrifilm Aerobic Count Plates, 3M Petrifilm Coliform Count Plates and 3M

Petrifilm Enterobacteriaceae Count Plates and incubated according to the manufacturer’s

1 World Bioproducts, Bothell, WA 2 3M and Petrifilm are trademarks of 3M, St. Paul, MN 3 VIDAS is a registered trademark of bioMerieux SA, Marcy I’Etoile 4 RapidChek is a registered trademark of Romer Labs, Inc., Union, MO 5 BAX is a registered trademark of E.I. du Pont de Nemours and Co., Wilmington, DE 6 ANSR is a trademark of Neogen Corporation, Lansing, MI 7 iQ-Check is a trade mark of BioRad Laboratories, Hercules, CA




specifications. For comparison, Butterfield’s Phosphate Buffer (“BPB”) solution was inoculated

with the same target organisms and 1 ml aliquots of the BPB were plated onto each type of 3M

Petrifilm Plates. Another test of HiCap Neutralizing Broth was performed to confirm that the

HiCap Neutralizing Broth itself does not cause a non-microbial reduction of the

triphenyltetrazolium chloride (“TTC”) indicator used in the 3M Petrifilm Plates. If such a

reduction occurs, the 3M Petrifilm Plates would show a pink or red color when compared to an

uninoculated plate. In these experiments, 1 ml aliquots of HiCap Neutralizing Broth (not

seeded with an organism) were added directly to 3M Petrifilm Plates and incubated according

to the manufacturer’s specifications.

Throughout this study, a pour plate procedure using Standard Methods Agar was utilized to

confirm inoculum levels.

4.0 MATERIALS AND METHODS

Pathogen Kit Compatibility Study: The following ATCC microorganisms were grown according

to Microbiologics’ recommended procedure1: Listeria ivanovii ATCC#19119, Listeria

monocytogenes ATCC#19114, Listeria monocytogenes ATCC#7644 and Listeria innocua

ATCC#33090; Salmonella enteritidis ATCC#13076, Salmonella Newport ATCC#6962, Salmonella

bongori ATCC#43975, Salmonella Abaetetuba ATCC#35640 and Salmonella Choleraesuis

ATCC#10708.

Overnight cultures of the Listeria and Salmonella strains were diluted to cell densities of

approximately 100-500 cells with the aid of a DensiCHEKTM

Plus instrument2, which provides

values in McFarland units proportional to microorganism density. To prepare an inoculum

cocktail of Listeria organisms, the diluted cultures were combined to give an approximately

equal concentration for each strain. A Salmonella inoculum cocktail was prepared in the same

way. Cell densities of each cocktail were determined using a pour plate procedure on Standard

Methods Agar, following the procedure described in the Compendium of Methods for the

Microbiological Examination of Foods 4th

Edition. The results of the pour plate tests gave an

average of 420 colony forming units per milliliter (cfu/ml) for the Listeria cocktail and 338

cfu/ml for the Salmonella cocktail.

Duplicate EZ Reach Samplers with polyurethane sponges hydrated with HiCap Neutralizing

Broth (World Bioproducts, Bothell, Washington, Item No. EZ-10HC-PUR) were inoculated with

0.1 ml of the Listeria cocktail, resulting in approximately 42 cells per sponge. Similarly,

duplicate EZ Reach Samplers with HiCap Neutralizing Broth were inoculated with 0.1 ml of the

Salmonella cocktail, resulting in approximately 34 cells per sponge. The inoculated EZ Reach

Samplers were then refrigerated at 4oC + 1

oC for 72 hours.

1 Microbiologics, St. Cloud, Minnesota Technical Information Bulletin TIB.081 Revision B 2 bioMerieux, Hazelwood, Missouri




After the 72 hour, the inoculated EZ Reach Samplers were enriched and tested according to the

protocol specified by the manufacturer of each pathogen kit. The pathogen kits included in the

study were VIDAS, RapidChek, BAX System Q7, ANSR and IQ Check. Also, HiCap Neutralizing

Broth, aseptically collected from each EZ Reach Samplers, was also tested directly in the

commercial test kits (without including an enrichment step) to confirm that the solution itself

does not cause interference.

3M Petrifilm Compatibility Study: The following ATCC microorganisms were grown according to

Microbiologics’ recommended procedure1: Staphylococcus aureus ATCC#25923, Escherichia coli

ATCC#25922, Shigella flexneri ATCC#12033. Each culture was then prepared as a suspension

using the DensiCHEK Plus.

HiCap Neutralizing Broth was aseptically collected from EZ Reach Samplers and dispensed into

sterile test tubes. HiCap was then inoculated with each test organisms to achieve a final

concentration of 10-50 cfu/ml. Butterfield’s Phosphate Buffer was also inoculated with each

test organism at the same cell concentration.

3M Petrifilm Aerobic Plate Count Plates were inoculated with either 1.0 ml of HiCap

Neutralizing Broth or 1.0 ml Butterfield’s Phosphate Buffer seeded with either S. aureus, E. coli,

or S. flexneri. 3M Petrifilm Coliform Count Plates were inoculated with either 1.0 ml of HiCap

Neutralizing Broth or 1.0 ml Butterfield’s Phosphate Buffer seeded with either E. coli or S.

flexneri. 3M Enterobacteriaceae Count Plates were inoculated with 1.0 ml of HiCap Neutralizing

or 1.0 ml Butterfield’s Phosphate Buffer seeded with either E. coli or S. flexneri.

To confirm cell concentrations in the HiCap and Butterfield’s solutions, a pour plate using

Standard Methods Agar was performed.

5.0 RESULTS

Table 1 shows the results of the HiCap Neutralizing Broth and pathogen kit compatibility study.

No inhibition was observed when HiCap Neutralizing Broth was tested directly in the

commercial pathogen kits or when the inoculated EZ Reach units were tested following the

protocols recommended by the manufacturer of each kit. The raw data results for each

pathogen kit are shown in Appendix 1.

1 Microbiologics, St. Cloud, Minnesota Technical Information Bulletin TIB.081 Revision B




Table 1. Compatibility of HiCap with Commercial Pathogen Kits.

Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2

HiCap + Listeria spp . + + + + + + + + + +

HiCap + Salmonella

spp.+ + + + + + + + + +

HiCap Only (Listeria

Assay)- - - - - - - - - -

HiCap Only

(Salmonella Assay)- - - - - - - - - -

VIDAS BAX Q7 BioRad IQ Check Neogen ANSR Romer RapidChekAssay Approach, in

Duplicate

Table 2 shows results when 3M Petrifilm Plates are inoculated with HiCap Neutralizing Broth

and Butterfield’s Phosphate Buffer seeded with various test organisms. Appendix 2 includes

photographs taken for detailed comparison of 3M Petrifilm Plates inoculated using HiCap

Neutralizing Broth and Butterfields Phosphate Buffer.

Table 2. HiCap Neutralizing Broth and Butterfield’s Phosphate Buffer with 3M Petrifilm Plates

Escherichia coli

Shigella flexneri

Staphylcoccus aureus bright pink colonies

Escherichia coli

Shigella flexneri

Escherichia coli

Shigella flexneri

Escherichia coli

Shigella flexneri

22

39

Butterfield's Phosphate BufferHiCap Neutralizing Broth

bright pink colonies

large bright pink

colonies, most had

gas

large bright pink

colonies, some had

gas

cfu/ml

32

32

19

26

NA

NA

Colony Description Colony Description

Assay Organism

NA

NA

cfu/ml

27

43

26

30

24

31

red colonies with gas

red colonies with no

gas

red colonies with gas

red-violet colonies

with no gas

APC Pour Plate

3M APC Petrifilm

3M Enterobacteriaceae

Petrifilm

3M Coliform Petrifilm

Shigella colonies with HiCap plated on

Enterobacteriaceae Petrifilm were lighter and

less developed at 24 hours than Butterfield's

Phosphate Buffer (BPB)

E. coli colonies with H iCap and BPB had similar

acid and gas production on

Enterobacteriaceae Petrifilm ; HiCap colonies

were less red than BPB colonies

E. coli colonies with H iCap and BPB had similar

gas production; BPB colonies were darker,

more violet in color and more defined

Shigella colonies w ith HiCap and BPB had

similar size; BPB colonies were darker red

pink colonies with

yellow acid and gas

pink, larger colonies

faint pink, small

colonies

19

22

16

34 27

Staphylcoccus aureus 18 NA 30 NA

36

pink colonies with

yellow acid and gas

pink colonies with

yellow acid and gas

pink colonies with

yellow acid and gas

Notes

Shigella colonies with HiCap were larger and

more pink in color

E. coli colonies with HiCap were larger and

brighter pink than BPB colonies; most colonies

had gas versus fewer colonies showing gas

with BPB

No difference noted

Any differences noted in Table 2 were made after close observations of colony appearance and

were not noted until the two films were placed side by side. All results conformed to the 3M

interpretation guide as typical colonies for morphology, color and gas production.




6.0 Conclusions

The solutions used in the collection of surface samples play a critical role in the success of an

environmental monitoring program. The solutions must be able to maintain microorganism

viability until the sample is processed, be appropriate for all laboratory tests that will be

performed on the collected sample, and have the capacity to neutralize sanitizers that are

typically used in the manufacturing environment that may be present on the sampled surface.

HiCap Neutralizing broth was evaluated for its appropriateness for use in an environmental

monitoring program. This study confirmed the ability of HiCap Neutralizing Broth to preserve

the viability of pathogenic microorganisms from collection to processing. Low levels of Listeria

and Salmonella species were preserved over a 72 hour period and detected using commercial

pathogen detection kits with no indication of interference by the HiCap Neutralizing Broth.

In addition, 3M Petrifilm Plates are widely used in the processing laboratory for quantification

and identification of microorganisms. When compared to Butterfield’s Phosphate Buffer,

HiCap Neutralizing Broth showed no differences in organism growth or colony development on

3M Petrifilm Plates.

7.0 Final Report Approval

Prepared by:

____________________________ 3-14-2013

Teri Ehlinger

Study Leader


Reviewed by:

____________________________ 3-14-2013

Joanne Ruebl

Study Manager





Appendix 1

Raw Data Results of HiCap Neutralizing Broth

with Commercial Pathogen Kits




VIDAS Assay Results

Sample

VIDAS RLU

Value – Rep 1

VIDAS RLU

Value – Rep 2

Threshold for

positive result

HiCap + Listeria spp. 1.68 1.61 ≥0.1

HiCap + Salmonella spp. 2.44 2.47 ≥0.23

HiCap Only – Listeria Assay 0.00 0.00 ≥0.1

HiCap Only – Salmonella Assay 0.05 0.04 ≥0.23




Salmonella Genus BAX Q7 Results




Listeria Genus BAX Q7 Results




Salmonella Genus iQ-Check Results




Listeria Genus iQ-Check Results




Salmonella Genus ANSR Results




Listeria Genus ANSR Results




Appendix 2

Pictures of 3M Petrifilm Plates after Incubation




Picture Series 1. 3M Petrifilm Aerobic Count Plates Inoculated with HiCap Neutralizing Broth

and Butterfield’s Phosphate Buffer




Picture Series 2. 3M Petrifilm Enterobacteriaceae Count Plates Inoculated with HiCap

Neutralizing Broth and Butterfield’s Phosphate Buffer




Picture Series 3. 3M Petrifilm Coliform Count Plates Inoculated with HiCap Neutralizing Broth

and Butterfield’s Phosphate Buffer

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