Performance Evaluation of HiCap TM Neutralizing Broth Monitoring/World...Performance Evaluation of...
Transcript of Performance Evaluation of HiCap TM Neutralizing Broth Monitoring/World...Performance Evaluation of...
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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
Cherney Microbiological Services, LTD
Study Sponsor World Bioproducts, LLC
PO Box 947 Bothell, WA 98041-0947
Tel: 877-260-6441; 425-242-4153
Cherney Microbiological Services, LTD Performance Evaluation of HiCapTM Neutralizing Broth
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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
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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
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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
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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
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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.
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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
Cherney Microbiological Services, LTD
Reviewed by:
____________________________ 3-14-2013
Joanne Ruebl
Study Manager
Cherney Microbiological Services, LTD
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Appendix 1
Raw Data Results of HiCap Neutralizing Broth
with Commercial Pathogen Kits
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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
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Salmonella Genus BAX Q7 Results
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Listeria Genus BAX Q7 Results
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Salmonella Genus iQ-Check Results
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Listeria Genus iQ-Check Results
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Salmonella Genus ANSR Results
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Listeria Genus ANSR Results
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Appendix 2
Pictures of 3M Petrifilm Plates after Incubation
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Picture Series 1. 3M Petrifilm Aerobic Count Plates Inoculated with HiCap Neutralizing Broth
and Butterfield’s Phosphate Buffer
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Picture Series 2. 3M Petrifilm Enterobacteriaceae Count Plates Inoculated with HiCap
Neutralizing Broth and Butterfield’s Phosphate Buffer
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Picture Series 3. 3M Petrifilm Coliform Count Plates Inoculated with HiCap Neutralizing Broth
and Butterfield’s Phosphate Buffer