Pathogens In Food Proficiency Testing Program Round...
Transcript of Pathogens In Food Proficiency Testing Program Round...
REPORT NO. 739
Pathogens In Food
Proficiency Testing Program
Round 24
November 2011
ACKNOWLEDGMENTS
PTA wishes to gratefully acknowledge the technical assistance provided for this program by Ms S Mott, Global Proficiency Ltd (New Zealand). This assistance included providing input into the design of the program, technical advice and discussion of the final report. PTA also wishes to gratefully acknowledge Global Proficiency Ltd (New Zealand) for producing the samples and Global Proficiency Pty Ltd (Australia) for distributing the samples.
© COPYRIGHT PROFICIENCY TESTING AUSTRALIA 2011
PO Box 7507 Silverwater NSW 2128 AUSTRALIA
TABLE OF CONTENTS
1. FOREWORD 1
2. FEATURES OF THE PROGRAM 1
3. FORMAT OF THE APPENDICES 1
4. DESIGN OF THE PROGRAM 2
5. HOMOGENEITY AND STABILITY TESTING 2
6. FALSE RESULTS 2
TABLE A: False Results 3
7. TECHNICAL COMMENTS 4
8. REFERENCES 6
APPENDICES
APPENDIX A
Summary of Results
Salmonella A1.1 - A1.2
Listeria A2.1 - A2.4
Staphylococcus aureus A3.1 - A3.2
APPENDIX B
Summary of Methods
Salmonella B1.1 - B1.2
Listeria B2.1 - B2.2
Staphylococcus aureus B3.1 - B3.2
APPENDIX C
Homogeneity and Stability Testing C1.1 - C1.3
APPENDIX D
Instructions to Participants D1.1
Results Sheets D2.1 - D2.4
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1. FOREWORD This report summarises the results of round twenty-four of a series of proficiency
testing programs involving the analysis of different food types for the detection of a range of pathogens.
Proficiency Testing Australia conducted the program in September / October
2011. The aim of the program was to assess laboratories’ abilities to competently perform the nominated tests.
The Program Coordinator was Dr M Bunt and the Technical Adviser was Ms S
Mott, Global Proficiency Ltd (New Zealand). This report was authorised by Ms F Ward, PTA Quality – Business Development Manager.
2. FEATURES OF THE PROGRAM (a) A total of eleven laboratories received samples, all of which returned results.
(b) The results reported by participants are presented in Appendix A.
(c) Laboratories were provided with five samples. Each sample consisted of a
freeze-dried vial with an accompanying matrix. Each matrix consisted of a total of 60 g of whole milk powder. Laboratories were also provided with "Instructions to Participants" and a "Results Sheet" (see Appendix D).
(d) Laboratories were requested to perform the tests according to their routine methods, with the Australian Standard method being preferred.
(e) Each laboratory was randomly allocated a unique code number for the program
to ensure confidentiality of results. Reference to each laboratory in this report is by its code number. Please note that one laboratory reported more than one set of results and, therefore, this laboratory’s code number (with letter) could appear several times in the same data set.
3. FORMAT OF THE APPENDICES
APPENDIX A Appendix A contains the results reported by participating laboratories for each of
the five samples. APPENDIX B Appendix B contains a summary of the methods used by each laboratory.
APPENDIX C Appendix C contains the results of the homogeneity and stability testing. APPENDIX D Appendix D contains the “Instructions to Participants” and pro-forma “Results
Sheets”.
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4. DESIGN OF THE PROGRAM Participants were asked to determine the presence or absence of Salmonella,
Listeria, Listeria monocytogenes (L. monocytogenes) and Staphylococcus aureus (S. aureus) in five samples of whole milk powder.
Each laboratory was provided with five samples labelled A, B, C, D and E and
was requested to test 25 grams of each sample for each analysis.
• Sample A contained Salmonella Derby. • Sample B was a negative sample. It did not contain Salmonella, Listeria or
Staphylococcus aureus.
• Sample C contained Salmonella Adelaide, Listeria innocua and Staphylococcus aureus.
• Sample D contained Salmonella Senftenberg, Listeria monocytogenes and
Staphylococcus aureus.
• Sample E contained Listeria monocytogenes and Staphylococcus aureus.
Other “typical” microflora were included in the samples (e.g. Escherichia coli, Enterococcus faecalis, etc.) The levels of Salmonella and Listeria in each sample were between 100 – 500 cfu per 25 g. The level of Staphylococcus aureus in each sample was approximately 10 000 cfu per 25 g. Microbiological samples for the Pathogens in Food program are manufactured according to Global Proficiency Ltd’s Standard Operating Procedures.
5. HOMOGENEITY AND STABILITY TESTING Prior to sample distribution, ten randomly selected samples from each matrix (A, B, C, D and E) were analysed for homogeneity by Global Proficiency Ltd (New Zealand). Based on the results of this testing, the homogeneity of the samples was established. Stability testing was also performed on the samples by Global Proficiency Ltd (New Zealand). The results showed that the samples were sufficiently stable for testing for the duration of the program. For more information on the homogeneity and stability testing, see Appendix C.
6. FALSE RESULTS Testing methods were pooled and results examined for laboratories reporting false positives and false negatives. The false positive and false negative results for this round of the program are summarised in the following table.
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TABLE A: FALSE RESULTS (by laboratory code number)
Presence / Absence of Salmonella in Whole Milk Powder
Sample False Positives False Negatives
A -
B 2, 5
C 2
D 2
E 5
Presence / Absence of Listeria in Whole Milk Powder
Sample False Positives False Negatives
A -
B 2
C -
D 2
E 2
Presence / Absence of Listeria monocytogenes in Whole Milk Powder
Sample False Positives False Negatives
A -
B 2
C -
D 2
E 2
Presence / Absence of Staphylococcus aureus in Whole Milk Powder
Sample False Positives False Negatives
A 5
B 5
C -
D -
E -
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7. TECHNICAL COMMENTS Response Rate All of the eleven laboratories that participated in the program submitted results for inclusion in the final report. All of the eleven participants reported results for Salmonella. Eight of the eleven participants (73%) reported results for Listeria. All of these eight laboratories also tested for Listeria monocytogenes. Eight of the eleven participants (73%) reported results for Staphylococcus aureus. Salmonella Results The results submitted by participants for Salmonella are presented in Appendix A1. Laboratory 2 reported a false positive result for sample B and false negative results for samples C and D. Laboratory 5 reported false positive results for samples B and E. Three of the eleven laboratories participating in the Salmonella test reported using immunoassay systems; two used the VIDAS® system, and one used the TECRA™ system. All three laboratories reported using Buffered Peptone Water as the Primary enrichment, and RVS or SX2 broths for the secondary enrichment, with XLD and Chromogenic agars used for the selective plating stage of the test. Confirmatory procedures included the use of rapid kits (API 20E and Microbact), Latex kits and serotyping, and traditional biochemical procedures. The remaining nine laboratories used cultural methods. All nine laboratories reported using Buffered Peptone Water as the Primary (non-selective) enrichment. For the secondary enrichment process, eight of the nine laboratories used RVS broth as one of the enrichments; four reported using MKTTn, two used M-broth and two used Selenite-Cystine. The Detection/Isolation stage of the test, using selective plating media, found that seven of the laboratories used XLD, with a range of other media used in conjunction. These included Chromogenic agar (four laboratories), BSA (four laboratories), BGA (four laboratories), Rambach (one laboratory) and MLCB (one laboratory). Confirmatory procedures used included traditional biochemical methods, serotyping techniques and rapid kits including API 20E and Microbact. Detailed method information for Salmonella is provided in Appendix B1.
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Listeria Results The results submitted by participants for Listeria and Listeria monocytogenes are presented in Appendix A2. Laboratory 2 reported a false positive result for sample B and false negative results for samples D and E for both Listeria and Listeria monocytogenes. In this round, four of the eight laboratories submitting results for Listeria testing reported using immunoassay systems; two used the VIDAS® system, one used TECRA™ and one did not state which system was used. The laboratory that used TECRA™ also ran the culture method concurrently and submitted results as a second report. One of the laboratories using VIDAS® also reported using the BAX® PCR system. Three laboratories reported using molecular biology techniques; specifically the BAX® PCR system. Three laboratories used cultural methods for the detection of Listeria and Listeria monocytogenes. The majority of participants reported using Oxford agar in conjunction with a second agar, which included PALCAM and Chromogenic agars. One laboratory stated that plating media were not used in the testing process. Confirmatory testing used a range of biochemical methods, haemolysis, the CAMP test and rapid kits including the API Listeria ID kit and Microbact 12L. Detailed method information for Listeria is provided in Appendix B2.
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Staphylococcus aureus Results The results submitted by participants for Staphylococcus aureus are presented in Appendix A3. Laboratory 5 reported false positive results for samples A and B. Of the eight laboratories submitting results for Staphylococcus aureus, one laboratory reported using the MPN technique with Modified Giolitti & Cantoni broth, with confirmation via tube coagulase from Baird-Parker agar. All other laboratories used the plating method with Baird-Parker agar. Five laboratories performed confirmatory testing using the tube coagulase test; one laboratory reported using Rabbit Plasma Fibrinogen agar, and one laboratory did not report undertaking confirmatory testing. Detailed method information for Staphylococcus aureus is provided in Appendix B3.
8. REFERENCES
1. Guide to Proficiency Testing Australia (2011). (This document is located on
the PTA website at www.pta.asn.au under Programs / Documents). 2. AS 5013.10: 2009 Food microbiology - Microbiology of food and animal
feeding stuffs - Horizontal method for the detection of Salmonella spp (ISO 6579:2002, MOD).
3. AS 5013.12.1: 2004 Food microbiology - Microbiology of food and animal
feeding stuffs - Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) - Technique using Baird-Parker agar medium.
4. AS 5013.24.1: 2009 Food microbiology - Microbiology of food and animal
feeding stuffs - Horizontal method for the detection and enumeration of Listeria monocytogenes - Detection method (ISO 11290-1:1996, MOD).
APPENDIX A
Summary of Results
Section A1
Salmonella
A1.1
Salmonella Results
Lab Code A B C D E False
Results 1A Present Absent Present Present Absent 1B Present Absent Present Present Absent 2 Present Present Absent Absent Absent 3 3 Present Absent Present Present Absent 4 Present Absent Present Present Absent 5 Present Present Present Present Present 2 6 Present Absent Present Present Absent 7 Present Absent Present Present Absent 8 Present Absent Present Present Absent 9 Present Absent Present Present Absent 10 Present Absent Present Present Absent 11 Present Absent Present Present Absent
Note: A highlighted result (i.e. bold print) is a false result and should be investigated.
A1.2
Salmonella Failure Rate
Sample No. of Results
A B C D E Total
False Results 0 2 1 1 1 5
Total Results 12 12 12 12 12 60
Failure rate (Sample A) = No. of False Results (A) Total No. of Results (A)
= 0 / 12
= 0%
Failure rate (Sample B) = No. of False Results (B) Total No. of Results (B)
= 2 / 12
= 16.7%
Failure rate (Sample C) = No. of False Results (C) Total No. of Results (C)
= 1 / 12
= 8.3%
Failure rate (Sample D) = No. of False Results (D) Total No. of Results (D)
= 1 / 12
= 8.3%
Failure rate (Sample E) = No. of False Results (E) Total No. of Results (E)
= 1 / 12
= 8.3%
Overall failure rate (Salmonella)
= Total No. of False Results Total No. of Results
= 5 / 60
= 8.3%
Section A2
Listeria
A2.1
Listeria Results
Lab Code A B C D E False
Results 1A Absent Absent Present Present Present 1B Absent Absent Present Present Present 2 Absent Present Present Absent Absent 3 3 Absent Absent Present Present Present 4 Absent Absent Present Present Present 6 Absent Absent Present Present Present 8 Absent Absent Present Present Present 9 Absent Absent Present Present Present 10 Absent Absent Present Present Present
Note: A highlighted result (i.e. bold print) is a false result and should be investigated.
A2.2
Listeria Failure Rate
Sample No. of Results
A B C D E Total
False Results 0 1 0 1 1 3
Total Results 9 9 9 9 9 45
Failure rate (Sample A) = No. of False Results (A) Total No. of Results (A)
= 0 / 9
= 0%
Failure rate (Sample B) = No. of False Results (B) Total No. of Results (B)
= 1 / 9
= 11.1%
Failure rate (Sample C) = No. of False Results (C) Total No. of Results (C)
= 0 / 9
= 0%
Failure rate (Sample D) = No. of False Results (D) Total No. of Results (D)
= 1 / 9
= 11.1%
Failure rate (Sample E) = No. of False Results (E) Total No. of Results (E)
= 1 / 9
= 11.1%
Overall failure rate (Listeria)
= Total No. of False Results Total No. of Results
= 3 / 45
= 6.7%
A2.3
Listeria monocytogenes Results
Lab Code A B C D E False
Results 1A Absent Absent Absent Present Present 1B Absent Absent Absent Present Present 2 Absent Present Absent Absent Absent 3 3 Absent Absent Absent Present Present 4 Not done Not done Absent Present Present 6 Absent Absent Absent Present Present 8 Absent Absent Absent Present Present 9 Absent Absent Absent Present Present 10 Absent Absent Absent Present Present
Note: A highlighted result (i.e. bold print) is a false result and should be investigated.
A2.4
Listeria monocytogenes Failure Rate
Sample No. of Results
A B C D E Total
False Results 0 1 0 1 1 3
Total Results 8 8 9 9 9 43
Failure rate (Sample A) = No. of False Results (A) Total No. of Results (A)
= 0 / 8
= 0%
Failure rate (Sample B) = No. of False Results (B) Total No. of Results (B)
= 1 / 8
= 12.5%
Failure rate (Sample C) = No. of False Results (C) Total No. of Results (C)
= 0 / 9
= 0%
Failure rate (Sample D) = No. of False Results (D) Total No. of Results (D)
= 1 / 9
= 11.1%
Failure rate (Sample E) = No. of False Results (E) Total No. of Results (E)
= 1 / 9
= 11.1%
Overall failure rate (L. monocytogenes)
= Total No. of False Results Total No. of Results
= 3 / 43
= 7.0%
Section A3
Staphylococcus aureus
A3.1
Staphylococcus aureus Results
Lab Code A B C D E False
Results 1A Absent Absent Present Present Present 1B Absent Absent Present Present Present 3 Absent Absent Present Present Present 4 Absent Absent Present Present Present 5 Present Present Present Present - 2 6 Absent Absent Present Present Present 8 Absent Absent Present Present Present 9 Absent Absent Present Present Present 10 Absent Absent Present Present Present
Note: A highlighted result (i.e. bold print) is a false result and should be investigated.
A3.2
Staphylococcus aureus Failure Rate
Sample No. of Results
A B C D E Total
False Results 1 1 0 0 0 2
Total Results 9 9 9 9 8 44
Failure rate (Sample A) = No. of False Results (A) Total No. of Results (A)
= 1 / 9
= 11.1%
Failure rate (Sample B) = No. of False Results (B) Total No. of Results (B)
= 1 / 9
= 11.1%
Failure rate (Sample C) = No. of False Results (C) Total No. of Results (C)
= 0 / 9
= 0%
Failure rate (Sample D) = No. of False Results (D) Total No. of Results (D)
= 0 / 9
= 0%
Failure rate (Sample E) = No. of False Results (E) Total No. of Results (E)
= 0 / 8
= 0%
Overall failure rate (S. aureus )
= Total No. of False Results Total No. of Results
= 2 / 44
= 4.5%
APPENDIX B
Summary of Methods
SECTION B1
Salmonella
B1.1
Salmonella – Media Lab
Code Non-selective Selective 1 Selective 2 Detection/
Isolation 1 Detection/ Isolation 2 Confirmation
1A Buffered peptone water SX2 broth Not done XLD Salmonella Chrom
Media Plate onto non-selective agar, Salmonella API kit
1B Buffered peptone water SX2 broth Not done XLD
Salmonella Chromogenic
Media Plate onto non-selective agar, Salmonella API kit
2 Buffered peptone water RVS broth Selenite Cystine Brilliant Green /
Phenol red agar BSA TSI, urea, L-lysine/LDC, β-galactosidase/ONPG, VP, indole, API 20E kit, O, Vi & H serotyping
3 Buffered peptone water MKTTn RVS broth XLD BSA In-house kit, O & H serotyping
4 Buffered peptone water RVS broth M-broth XLD, Salmonella
Chromogenic agar BSA Plate onto non-selective agar, Vitek ID kit, O & H serotyping
5 Buffered peptone water RVS broth MKTTn broth XLD BGA Plate onto non-selective agar, TSI, urea, L-lysine/LDC,
indole
6 Buffered peptone water RVS broth Not done Tecra Ultima XLD, Chromogenic
Salmonella agar Microbact 12E & latex
7 Buffered peptone water M-broth MSC broth, RVS
broth XLD, BSA, Rambach
XLD, BSA, Rambach Microbact (Oxoid) kit
8 Buffered peptone water RVS broth Selenite Cystine
broth BSA BGA (Brilliant
Green phenol red agar)
Plate onto non-selective agar, API 20E kit, O, Vi & H serotyping
9 Buffered peptone water RVS broth Not done XLD, Vidas SLM Chrom ID Plate onto non-selective agar, L-lysine/LDC, β-
galactosidase/ONPG, API 20E kit, O, Vi & H serotyping
10 Buffered peptone water MKTTn RVS broth XLD BGA Plate onto non-selective agar, TSI, urea, L-lysine/LDC, β-
galactosidase/ONPG, VP, indole, O & H serotyping
11 Buffered peptone water MKTTn RVS broth XLD
Salmonella Chromogenic Agar,
MLCB API 20E kit, O & H serotyping
B1.2
Salmonella – Incubation Temperature ( °°°°C) Salmonella – Duration of Incubation (hours) Lab
Code Non-selective Selective 1 Selective 2 Detection/
Isolation 1 Detection/ Isolation 2 Confirm. Non-
selective Selective 1 Selective 2 Detection/ Isolation 1
Detection/ Isolation 2 Confirm.
1A 37 42 37 24 24 48 24
1B 37 42 37 37 37 24 24 24 48 24
2 37 41.5 37 41.5 37 37 24 48 24 48 48 24
3 37 37 42 37 37 37 18 24 24 24 24, 48 24
4 37 42 42 37 35 37 18 18, 6 18 24 24, 48 18
5 37 42 37 37 37 37 18 24 24 24 24 24
6 37 42 37 24 24 24, 48
7 37 37 37, 42 37 37 37 18 18 24 48 48 18
8 37 42 37 37 37 37 18 24 24 48 24 24
9 37 42 37 37 18 18 18 18
10 37 37 42 37 37 37 18 24 24 24 24 24
11 37 37 42 37 37 37 24 24 24 24 24 18
SECTION B2
Listeria
B2.1
Listeria – Media Lab
Code Non-selective Primary Second
Selective Agar Plating 1 Agar Plating 2 Other Detection Confirmation
1A Not done Half Fraser broth
Full strength Fraser broth
Oxford agar, Chromogenic
agar
PALCAM agar, Chromogenic
agar Not done Plate onto non-selective agar, motility test, Listeria Microgen
ID kit, Gram stain, catalase test
1B Not done Half Fraser broth
Full strength Fraser broth
Oxford agar, Chromogenic
agar
PALCAM agar, Chromogenic
agar Not done Plate onto non-selective agar, motility test, Listeria Microgen
ID kit, Gram stain, catalase test
2
Buffered Listeria
enrichment broth
PALCAM agar API Listeria Strip Elisa kit Motility test, Gram stain, catalase test, Blood agar
3 Not done Half Fraser broth
Full strength Fraser broth
Oxford agar, Listeria
selective agar
Chromogenic agar (Oxoid
Brilliance Listeria)
BAX PCR Listeria
monocytogenes
Plate onto non-selective agar, β haemolysis, serological test, Listeria API ID kit
4 Half Fraser broth
Full strength Fraser broth
Oxford agar, Chromogenic
agar (Chromocult Listeria agar)
PALCAM agar Vidas Elisa kit,
BAX DNA probe
Plate onto non-selective agar, Gram stain, catalase test
6.1 Not done
Buffered Listeria
enrichment broth
Full strength Fraser broth Not done Not done Tecra VIA Elisa
kit Plate onto non-selective agar, β haemolysis, CAMP test, motility test, Microbact 12L ID kit
6.2 Not done
Buffered Listeria
enrichment broth
Not done Oxford agar Not done Not done Plate onto non-selective agar, β haemolysis, CAMP test, motility test, Microbact 12L ID kit
8 Not done Half Fraser broth
MOPS - BLEB for BAX Oxford agar PALCAM agar BAX DNA
probe Plate onto non-selective agar, β haemolysis, CAMP test, motility test, sugar fermentation tests, BAX DNA probe
9 Half Fraser broth
Full strength Fraser broth
Chromogenic agar (OAA) Vidas Lis Elisa
kit Plate onto non-selective agar, β haemolysis, motility test, API Lis ID kit
10 Not done Half Fraser broth
Full strength Fraser broth
Oxford agar, Chromogenic agar (ALOA)
PALCAM agar Not done Plate onto non-selective agar, β haemolysis, CAMP test, motility test, sugar fermentation tests
B2.2
Listeria – Incubation Temperature ( °°°°C) Listeria – Duration of Incubation (hours) Lab
Code Non-selective Primary Second
Selective Agar
Plating 1 Agar
Plating 2 Other
Detection Confirm. Non-selective Primary Second
Selective Agar
Plating 1 Agar
Plating 2 Other
Detection Confirm.
1A 30 30 37 37 30 24 24 48 48 24
1B 30 30 37 37 30 24 24 48 48 24
2 30 35 35 30, 35 48 48 24 24
3 30 37 37 37 37 24 48 48 48 24
4 30 30 30, 37 30, 37 37 22 22 24
6.1 30 30 24 24
6.2 30 37 48 48
8 30 35 37 37 25, 37 24 24 48 48 24, 48, 7 days
- sugar fermentation
9 30 30 37 37 24 24 24 24
10 30 37 37 37 37 24 48 48 48 24, sugar
fermentation: up to 7 days
Notes: 1. Laboratory 6.1 method information is for samples A, B, C and D. 2. Laboratory 6.2 method information is for sample E only.
SECTION B3
Staphylococcus aureus
B3.1
Staphylococcus aureus – Media Staphylococcus aureus – Plating / Inoculation Details Lab
Code Plating Method Plating Confirmation MPN Detection MPN Confirmation Plating Method MPN Detection
1A Baird-Parker Tube coagulase, BHIB → rabbit plasma Not done Not done 0.1 mL per plate
1B Baird-Parker Tube coagulase, BHIB → rabbit plasma Not done Not done 0.1 mL per plate
3 Baird-Parker Tube coagulase Not done Not done 1 mL over 3 plates, 0.1 mL per plate
4 Baird-Parker Rabbit Plasma Fibrinogen agar Not done Not done 1 mL over 3 plates
5 Baird-Parker Tube coagulase Not done Not done 0.1 mL per plate
6 Not done Not done Modified Giolitti & Cantoni broth
Baird-Parker, tube coagulase 1 g (10 mL of 10-1)
8 Baird-Parker Not done Not done Not done 1 mL over 3 plates
9 Baird-Parker Tube coagulase 1 mL over 3 plates
10 Baird-Parker Tube coagulase Not done 1 mL over 3 plates
B3.2
Staphylococcus aureus – Incubation Temperature ( °°°°C) Staphylococcus aureus – Duration of Incubation (hours) Lab
Code Plating Method Plating Confirm. MPN Detection MPN Confirm. Plating Method Plating Confirm. MPN Detection MPN Confirm.
1A 37 35-37 48 24
1B 37 35-37 48 24
3 37 35-37 48 4
4 37 35-37 48 24-48
5 37 35-37 48 4
6 37 37 48 48
8 37 48
9 37 35-37 48 4
10 37 35-37 48 4, 24
APPENDIX C
Homogeneity and
Stability Testing
C1.1
HOMOGENEITY TESTING RESULTS
Ten samples from each matrix (A, B, C, D and E) were randomly chosen and tested by Global Proficiency Ltd (New Zealand) to confirm that the samples were homogeneous. Homogeneity testing was performed on 17 August 2011. The results were analysed prior to sample dispatch. For Salmonella, the method of testing was enumeration via spread plate technique using XLD agar. The samples were verified using AS 5013.10: 2009 (ISO equivalent – ISO 6579: 2002). For Listeria, the method of testing was enumeration via spread plate technique using PALCAM agar. The samples were verified using AS 5013.24.1: 2009 (ISO equivalent – ISO 11290-1: 1996, MOD). For Staphylococcus aureus, the method of testing was enumeration via spread plate technique using Baird-Parker agar. The samples were verified using AS 5013.12.1: 2004. The results are tabulated below.
Sample A (containing Salmonella Derby)
Sample Salmonella Listeria L. monocytogenes S. aureus 6 Detected Not detected Not detected Not detected 20 Detected Not detected Not detected Not detected 21 Detected Not detected Not detected Not detected 24 Detected Not detected Not detected Not detected 26 Detected Not detected Not detected Not detected 27 Detected Not detected Not detected Not detected 44 Detected Not detected Not detected Not detected 48 Detected Not detected Not detected Not detected 72 Detected Not detected Not detected Not detected 79 Detected Not detected Not detected Not detected
Sample B (negative sample)
Sample Salmonella Listeria L. monocytogenes S. aureus 1 Not detected Not detected Not detected Not detected 2 Not detected Not detected Not detected Not detected 3 Not detected Not detected Not detected Not detected 8 Not detected Not detected Not detected Not detected 10 Not detected Not detected Not detected Not detected 32 Not detected Not detected Not detected Not detected 34 Not detected Not detected Not detected Not detected 36 Not detected Not detected Not detected Not detected 41 Not detected Not detected Not detected Not detected 60 Not detected Not detected Not detected Not detected
C1.2
Sample C (containing Salmonella Adelaide, Listeria innocua and S. aureus)
Sample Salmonella Listeria L. monocytogenes S. aureus 23 Detected Detected Not detected Detected 28 Detected Detected Not detected Detected 37 Detected Detected Not detected Detected 54 Detected Detected Not detected Detected 55 Detected Detected Not detected Detected 56 Detected Detected Not detected Detected 57 Detected Detected Not detected Detected 59 Detected Detected Not detected Detected 62 Detected Detected Not detected Detected 66 Detected Detected Not detected Detected
Sample D (containing Salmonella Senftenberg, L. monocytogenes and S. aureus)
Sample Salmonella Listeria L. monocytogenes S. aureus 4 Detected Detected Detected Detected 5 Detected Detected Detected Detected 6 Detected Detected Detected Detected 18 Detected Detected Detected Detected 39 Detected Detected Detected Detected 49 Detected Detected Detected Detected 53 Detected Detected Detected Detected 67 Detected Detected Detected Detected 68 Detected Detected Detected Detected 69 Detected Detected Detected Detected
Sample E (containing L. monocytogenes and S. aureus)
Sample Salmonella Listeria L. monocytogenes S. aureus 16 Not detected Detected Detected Detected 24 Not detected Detected Detected Detected 30 Not detected Detected Detected Detected 33 Not detected Detected Detected Detected 35 Not detected Detected Detected Detected 61 Not detected Detected Detected Detected 65 Not detected Detected Detected Detected 68 Not detected Detected Detected Detected 77 Not detected Detected Detected Detected 79 Not detected Detected Detected Detected
Based on the above testing results, the homogeneity of the samples was established.
C1.3
STABILITY TESTING RESULTS To determine whether the samples used for this program were stable, three samples from each matrix (A, B, C, D and E) were randomly chosen and tested by Global Proficiency Ltd (New Zealand). The stability testing took place on 29 September 2011. The results are tabulated below.
Sample A (containing Salmonella Derby)
Sample Salmonella Listeria L. monocytogenes S. aureus 42 Detected Not detected Not detected Not detected 63 Detected Not detected Not detected Not detected 66 Detected Not detected Not detected Not detected
Sample B (negative sample)
Sample Salmonella Listeria L. monocytogenes S. aureus 6 Not detected Not detected Not detected Not detected 22 Not detected Not detected Not detected Not detected 46 Not detected Not detected Not detected Not detected
Sample C (containing Salmonella Adelaide, Listeria innocua and S. aureus)
Sample Salmonella Listeria L. monocytogenes S. aureus 29 Detected Detected Not detected Detected 40 Detected Detected Not detected Detected 61 Detected Detected Not detected Detected
Sample D (containing Salmonella Senftenberg, L. monocytogenes and S. aureus)
Sample Salmonella Listeria L. monocytogenes S. aureus 13 Detected Detected Detected Detected 29 Detected Detected Detected Detected 63 Detected Detected Detected Detected
Sample E (containing L. monocytogenes and S. aureus)
Sample Salmonella Listeria L. monocytogenes S. aureus 21 Not detected Detected Detected Detected 36 Not detected Detected Detected Detected 41 Not detected Detected Detected Detected
Based on these results, the samples were considered to be stable during the period that this proficiency testing program was conducted.
APPENDIX D
Instructions to Participants
and
Results Sheets
D1.1
Pathogens in Food 24 September 2011 Page 1 of 5
PROFICIENCY TESTING AUSTRALIA PATHOGENS IN FOOD PROGRAM – ROUND 24
INSTRUCTIONS TO PARTICIPANTS
On receipt of samples: Open the container immediately and check the contents are in order • Record the temperature of the samples. • Return the contents to the original packaging. • Transfer the samples to a refrigerator (2-5 °C) for storage prior to testing. • Protect the samples from light. Prior to testing please note: • Five samples (labelled A, B, C, D, E) each containing 60 g of whole milk powder are to be tested for the
presence/absence of Salmonella and Listeria as per instructions below. Testing for the presence/absence of Staphylococcus aureus is also offered in this round. If you have this capability, you are encouraged to perform the test.
• Samples are for laboratory use only. • Store your samples in the original packaging between 2-5 °C until testing commences. • It is strongly recommended that testing is initiated within 48 hours of receipt of the samples. • Where practical your laboratory is encouraged to test different samples using different analysts. • Laboratories should perform all testing using their routine test methods. • Listeria speciation is not mandatory, but is encouraged. • Salmonella serotyping is not required. • Confirmatory testing for S. aureus is required. • Your laboratory has been allocated the code number shown on the attached Results and Method
Information Sheets. Instructions You have been supplied with freeze dried vials and accompanying whole milk powder matrices in foil laminate sachets. Please find below instructions for the re-hydration and preparation of the freeze-dried vials and steps for the preparation of the matrix. 1. Re-hydrate each freeze-dried vial by adding 3.0 mL of sterile diluent (e.g. 0.1% (w/v) peptone and 0.85%
(w/v) NaCl (ISO, 6887-1)) at room temperature. 2. Allow standing at room temperature for 10 minutes. 3. Mix the vial contents using a vortex mixer for 15 seconds. 4. Aseptically open the sachets. Weigh out 25 g for each of the Salmonella and Listeria tests to be
performed. Add 225 mL enrichment broth. Mix to dissolve the milk powder. Add 1 mL of the rehydrated vial contents and continue as per your Standard method. Please note: S. aureus testing: Aliquots are to be removed from the initial non-selective Salmonella enrichment once prepared. If following the plating procedure for S. aureus, levels have been designed to suit the inoculum volume of 1 mL (over 3 plates) if following AS 5013.12.1 – 2004.
5. Proceed as per your laboratory test method. The Australian Standard Methods are the preferred test methods.
6. Report results as presence or absence per 25 gram of sample in Table A of the supplied Results Sheets by filling in (•) in the appropriate circles. If Salmonella, Listeria or S. aureus are not detected in a sample then this should be indicated by filling in (•) in the circle alongside “absent”.
7. Report all method information in Tables B, C and D of the supplied Results Sheets by filling in (•) in the appropriate circles. If more than one method is used for a test report each result separately (copy and use a separate Results Sheet for each method).
Please return results no later than Tuesday 11 October 2011 to: Mark Bunt Proficiency Testing Australia PO Box 7507 Telephone: +61 2 9736 8397 (1300 782 867) Silverwater NSW 2128 Fax: +61 2 9743 6664 AUSTRALIA Email: [email protected]
Pathogens in Food 24 September 2011 Page 2 of 5
PTA Pathogens in Food (Round 24) Proficiency Testin g Program
RESULTS SHEET
Date samples arrived Sample temperature Date testing began Signature
Laboratory
Code:
Table A: Results
Test Sample A Sample B Sample C Sample D Sample E
Salmonella � Present
� Absent
� Not done
� Present
� Absent
� Not done
� Present
� Absent
� Not done
� Present
� Absent
� Not done
� Present
� Absent
� Not done
Listeria � Present
� Absent
� Not done
� Present
� Absent
� Not done
� Present
� Absent
� Not done
� Present
� Absent
� Not done
� Present
� Absent
� Not done
Listeria
monocytogenes
� Present
� Absent
� Not done
� Present
� Absent
� Not done
� Present
� Absent
� Not done
� Present
� Absent
� Not done
� Present
� Absent
� Not done
Staphylococcus
aureus
� Present
� Absent
� Not done
� Present
� Absent
� Not done
� Present
� Absent
� Not done
� Present
� Absent
� Not done
� Present
� Absent
� Not done
D2.1
Pathogens in Food 24 September 2011 Page 3 of 5
Table B: Method Information: Salmonella Laboratory Code Please indicate the methodology used by filling in (•) in the appropriate circles below:
Method Non-selective Enrichment
Selective Enrichment Medium 1
Selective Enrichment Medium 2
Detection/Isolation Confirmation of suspect Salmonella
Media
� Not done
� Buffered
peptone water
� Other (specify)
______________
� Not done
� MSC broth
� RVS broth
� MKTTn
� M-broth
� Other
(specify)
_____________
� Not done
� MSC broth
� RVS broth
� MKTTn
� M-broth
� Other
(specify)
_____________
� Not done PLATING Medium 1
� XLD
� BSA
� Other (specify)
_____________
� Not done PLATING Medium 2
� XLD
� BSA
� Other (specify)
________________
� Not done � Plate onto non-selective
agar BIOCHEMICAL
� TSI � Urea � L-lysine / LDC � β-galactosidase/ ONPG � VP � Indole � Commercial kit (specify) ________________ SEROTYPING
� O � Vi � H � Commercial ID kit (specify) ________________ � Other (specify) __________________
Incubation Temperature
� 35 ºC � 37 ºC � 42 ºC � Other ____ ºC
� 35 ºC � 37 ºC � 42 ºC � Other ____ ºC
� 35 ºC � 37 ºC � 42 ºC � Other ____ ºC
� 35 ºC � 37 ºC � 42 ºC � Other _____ ºC
� 35 ºC � 37 ºC � 42 ºC � Other _____ ºC
� 35 ºC � 37 ºC � 42 ºC � Other _____ ºC
Duration of Incubation
� 18 hours � 24 hours � 48 hours � 72 hours � Other_______
� 18 hours � 24 hours � 48 hours � 72 hours � Other_______
� 18 hours � 24 hours � 48 hours � 72 hours � Other_______
� 18 hours � 24 hours � 48 hours � 72 hours � Other________
� 18 hours � 24 hours � 48 hours � 72 hours � Other________
� 18 hours � 24 hours � 48 hours � 72 hours � Other __________
D
2.2
Pathogens in Food 24 September 2011 Page 4 of 5
Table C: Method Information: Listeria Laboratory Code Please indicate the methodology used by filling in (•) in the appropriate circles below:
Method Non-selective Enrichment
Primary Selective
Enrichment
Secondary Selective
Enrichment
Selective Agar Plating
Medium 1 Medium 2
Other Detection Methods
Confirmation of suspect Listeria
Media
� Not done � Tryptone soy broth with yeast extract � Other (specify) _____________
� Not done � Buffered Listeria enrichment broth � Half Fraser broth � UVM-I � Other (specify) _____________
� Not done � Full strength Fraser broth � UVM-II � Other (specify) _____________
� Not done � Oxford agar � Chromogenic agar (specify)
_____________
� Other agar (specify) _____________
� Not done � PALCAM agar � Chromogenic agar (specify)
_____________
� Other agar (specify) _____________
� Not done � Elisa kit (specify type)
_____________
� DNA probe (specify type)
_____________ � Other detection methods (specify type) _____________
� Not done � Plate onto non-selective agar � β haemolysis � CAMP test � Motility test � Growth in anaerobic conditions � Sugar fermentation tests � Latex test � DNA probe � Serological test � Commercial ID kit (specify) _____________ � Other (specify) _____________
Incubation Temperature
� 25 ºC � 30 ºC � 37 ºC � Other _____ ºC
� 25 ºC � 30 ºC � 37 ºC � Other _____ ºC
� 25 ºC � 30 ºC � 37 ºC � Other _____ ºC
� 25 ºC � 30 ºC � 37 ºC � Other _____ ºC
� 25 ºC � 30 ºC � 37 ºC � Other _____ ºC
� 25 ºC � 30 ºC � 37 ºC � Other _____ ºC
� 25 ºC � 30 ºC � 37 ºC � Other _____ ºC
Duration of Incubation
� 4 hours � 24 hours � 48 hours � Other __________
� 24 hours � 48 hours � 72 hours � Other __________
� 24 hours � 48 hours � 72 hours � Other __________
� 24 hours � 48 hours � 72 hours � Other __________
� 24 hours � 48 hours � 72 hours � Other __________
� 24 hours � 48 hours � 72 hours � Other __________
� 24 hours � 48 hours � 72 hours � Other __________
D2.3
Pathogens in Food 24 September 2011 Page 5 of 5
Table D: Method Information: Staphylococcus aureus (Presence/Absence) Laboratory Code Please indicate the methodology used by filling in (•) in the appropriate circles below:
Method Plating Method Confirmation from Plating Method
Most Probable Number Method: Detection
Confirmation from MPN Detection Method
Media
� Not done
� Baird-Parker
� Rabbit Plasma
Fibrinogen agar
� Other (specify)
___________________
� Not done
� Tube Coagulase
� Thermonuclease test*
� Latex Agglutination
(specify type)
___________________
� Other (specify)
___________________
� Not done
� Modified Giolitti &
Cantoni broth
� Trypticase Soy broth
� Other (specify)
___________________
� Not done
� Baird-Parker
� Rabbit Plasma
Fibrinogen agar
� Tube Coagulase
� Latex Agglutination
(specify type)
___________________
� Other (specify)
___________________ Plating / Inoculation Details
���� 1 mL over 3 plates (recommended) � 0.1 mL per plate � Other (specify)
______________
� 1 g (10 mL of 10-1) � 0.1 g (1 mL of 10-1) � 0.01 g (1 mL of 10-2) � Other (specify)
______________
Incubation Temperature
� 35 ºC � 37 ºC � Other ____ ºC
* � 35–37 ºC � 50 ºC � Other ____ ºC
� 35 ºC � 37 ºC � Other ____ ºC
� 35 ºC � 37 ºC � Other ____ ºC
Duration of Incubation
� 24 hours � 45 hours � 48 hours � Other_______
* � 2 hours � 4 hours � Other_______
� 24 hours � 48 hours � Other __________
� 24 hours � 45 hours � 48 hours � Other_______
D
2.4
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