Pathogens in Food Proficiency Testing Program Round 19capeyork.myownserver.net/~ptaasnau/documents/613.pdf · TABLE OF CONTENTS 1. FOREWORD 1 2. FEATURES OF THE PROGRAM 1 3. FORMAT

REPORT NO. 613

Pathogens in Food

Proficiency Testing Program

Round 19

June 2009

ACKNOWLEDGMENTS

PTA wishes to gratefully acknowledge the technical assistance provided for this program by Ms S Mott, AsureQuality Limited (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 AsureQuality Limited (New Zealand) for producing the samples and AsureQuality Limited (Australia) for distributing the samples.

© COPYRIGHT PROFICIENCY TESTING AUSTRALIA 2009

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

Bacillus cereus A3.1 - A3.2

APPENDIX B

Summary of Methods

Salmonella B1.1 - B1.4

Listeria B2.1 - B2.2

Bacillus cereus B3.1

APPENDIX C

Homogeneity Testing C1.1 - C1.2

Stability Testing 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 nineteen 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 April / May 2009. The

Program Coordinator was Dr M Bunt. The aim of the program was to assess laboratories' ability to competently perform the nominated tests.

2. FEATURES OF THE PROGRAM (a) A total of eighteen laboratories received samples, one of which did not return

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 rice flour. 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 Bacillus cereus (B. cereus) in five samples of rice flour.

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 Listeria monocytogenes. • Sample B contained Salmonella Adelaide and Bacillus cereus.

• Sample C contained Salmonella Derby and Listeria innocua.

• Sample D contained Bacillus cereus.

• Sample E contained Salmonella Senftenberg (H2S negative strain) and

Listeria monocytogenes.

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 Bacillus cereus in each sample was approximately 10 000 cfu per 25 g.

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 AsureQuality Limited (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 AsureQuality Limited (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 Rice Flour

Sample False Positives False Negatives

A 9

B -

C -

D 9

E 11

Presence / Absence of Listeria in Rice Flour


A 11

B 12

C -

D 1, 11, 12

E 11

Presence / Absence of Listeria monocytogenes in Rice Flour


A 15A, 15B

B -

C 3

D -

E 15A, 15B

Presence / Absence of Bacillus cereus in Rice Flour


A -

B -

C -

D 11

E -

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7. TECHNICAL COMMENTS Response Rate Of the eighteen laboratories that participated in the program, seventeen (94%) submitted results for inclusion in the final report. All of the seventeen participants that submitted results for the program reported results for Salmonella. Fifteen of the seventeen participants that submitted results for the program (88%) reported results for Listeria. Fourteen of these fifteen laboratories (93%) also tested for Listeria monocytogenes. Eleven of the seventeen participants that submitted results for the program (65%) reported results for Bacillus cereus. One laboratory (15) submitted more than one set of results. Salmonella Results Of the seventeen participants that submitted results for Salmonella, two submitted false results. Laboratory 9 reported false positive results for samples A and D, which did not contain Salmonella. Laboratory 11 reported a false negative result for sample E, which contained an H2S negative strain of Salmonella Senftenberg. The failure rates for the samples for Salmonella are calculated in Appendix A. The overall failure rate for Salmonella is also calculated in Appendix A. Here it was found that samples A, D and E had the highest failure rate of all the samples for Salmonella (5.6%). There were no false results reported for samples B and C. The overall failure rate for Salmonella was found to be 3.3%. Listeria Results Of the fifteen participants that submitted results for Listeria, three submitted false results. Laboratory 11 did not detect Listeria in samples A and E but did correctly report the presence of Listeria monocytogenes in samples A and E. Therefore, it is possible that laboratory 11 misinterpreted “Listeria” in Table A of the Results Sheets to mean Listeria species other than Listeria monocytogenes. Laboratory 11 also incorrectly detected the presence of Listeria in sample D, as did laboratories 1 and 12. Laboratory 12 also incorrectly detected the presence of Listeria in sample B. The failure rates for the samples for Listeria are calculated in Appendix A. The overall failure rate for Listeria is also calculated in Appendix A. Here it was found that sample D had the highest failure rate of all the samples for Listeria (18.8%), followed by samples A, B and E (6.3%). There were no false results reported for sample C. The overall failure rate for Listeria was found to be 7.5%.

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Listeria monocytogenes Results Of the fourteen participants that tested the samples for Listeria monocytogenes, two submitted false results. Laboratory 15 submitted two sets of results (15A and 15B). Both of these sets of results incorrectly indicated that Listeria monocytogenes was not present in samples A and E. Laboratory 3 incorrectly indicated that Listeria monocytogenes was present in sample C, which actually contained Listeria innocua. Laboratory 12, which reported false positive results for Listeria for samples B and D, did not test the samples for Listeria monocytogenes. Laboratories 1 and 11, which reported false positives results for Listeria for sample D, correctly did not detect Listeria monocytogenes in sample D. The failure rates for the samples for Listeria monocytogenes are calculated in Appendix A. The overall failure rate for Listeria monocytogenes is also calculated in Appendix A. Here it was found that samples A and E had the highest failure rate of all the samples for Listeria monocytogenes (13.3%), followed by sample C (6.7%). There were no false results reported for samples B and D. The overall failure rate for Listeria monocytogenes was found to be 7.0%. Bacillus cereus Results Of the eleven participants that submitted results for Bacillus cereus, one submitted a false result. Laboratory 11 reported a false negative result for sample D. The failure rates for the samples for Bacillus cereus are calculated in Appendix A. The overall failure rate for Bacillus cereus is also calculated in Appendix A. The failure rate for sample D was found to be 8.3%, while the overall failure rate for Bacillus cereus was found to be 1.7%.

Method Commentary Salmonella Sixteen out of seventeen laboratories used buffered peptone water as the non-selective enrichment; thirteen used RVS broth in conjunction with another broth (eleven used MKTTn as the second broth) for the selective enrichment stage. Other participants used broths specific for the immunoassay systems used or those prescribed by other methods. Eleven out of seventeen laboratories reported using cultural methods for the detection of Salmonella. Several laboratories reported using immunoassay systems and one laboratory reported using BAX-PCR.

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There were a range of different confirmatory procedures used including traditional biochemical methods, serotyping techniques and a range of rapid kits. Detailed method information is provided in Appendix B1. Listeria Thirteen out of fifteen laboratories reported using Half Fraser broth for the primary enrichment stage; thirteen reported using full strength Fraser broth for the secondary enrichment stage. Eight laboratories reported that immunoassay systems were used for the detection of Listeria / Listeria monocytogenes, with confirmation using a range of both biochemical methods, haemolysis (including CAMP) and rapid kits. Four laboratories reported using BAX-PCR followed by confirmation as detailed above. Other participants reported cultural techniques followed by biochemical methods, haemolysis, the CAMP test and rapid kits. Detailed method information is provided in Appendix B2. Bacillus cereus Seven out of eleven laboratories reported using MYP agar to select / differentiate Bacillus cereus in the proficiency samples. Two laboratories used PEMBA agar and one laboratory reported using BCSA agar to detect the organism. Another laboratory used a Most Probable Number (MPN) method using Trypticase Soy Polymixin broth. All laboratories performed a confirmation step; three performed spore and lipid stains, two examined haemolysis, one used an API system and all other laboratories used a combination of spore and lipid stains / haemolysis / Gram stain. Detailed method information is provided in Appendix B3.

8. REFERENCES

1. Guide to Proficiency Testing Australia (2008). (This document is located on

the PTA website at www.pta.asn.au under Programs / Documents). 2. AS 5013.2: 2007 Food microbiology - Microbiology of food and animal

feeding stuffs - Horizontal method for the enumeration of Bacillus cereus - Colony-count technique at 30C (ISO 7932:2004, MOD).

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3. AS 5013.10: 2004 Food microbiology - Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp.

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 1 Absent Present Present Absent Present 2 Absent Present Present Absent Present 3 Absent Present Present Absent Present 4 Absent Present Present Absent Present 5 Absent Present Present Absent Present 6 Absent Present Present Absent Present 7 Absent Present Present Absent Present 8 Absent Present Present Absent Present 9 Present Present Present Present Present 2

11 Absent Present Present Absent Absent 1 12 Absent Present Present Absent Present 13 Absent Present Present Absent Present 14 Absent Present Present Absent Present

15A Absent Present Present Absent Present 15B Absent Present Present Absent Present 16 Absent Present Present Absent Present 17 Absent Present Present Absent Present 18 Absent Present Present Absent Present

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 1 0 0 1 1 3

Total Results 18 18 18 18 18 90

Failure rate (Sample A) = No. of False Results (A) Total No. of Results (A)

= 1 / 18

= 5.6%

Failure rate (Sample B) = No. of False Results (B) Total No. of Results (B)

= 0 / 18

= 0%

Failure rate (Sample C) = No. of False Results (C) Total No. of Results (C)

= 0 / 18

= 0%

Failure rate (Sample D) = No. of False Results (D) Total No. of Results (D)

= 1 / 18

= 5.6%

Failure rate (Sample E) = No. of False Results (E) Total No. of Results (E)

= 1 / 18

= 5.6%

Overall failure rate (Salmonella)

= Total No. of False Results Total No. of Results

= 3 / 90

= 3.3%

Section A2

Listeria

A2.1

Listeria Results


Results 1 Present Absent Present Present Present 1 2 Present Absent Present Absent Present 3 Present Absent Present Absent Present 4 Present Absent Present Absent Present 5 Present Absent Present Absent Present 6 Present Absent Present Absent Present 7 Present Absent Present Absent Present 9 Present Absent Present Absent Present

11 Absent Absent Present Present Absent 3 12 Present Present Present Present Present 2 13 Present Absent Present Absent Present

15A Present Absent Present Absent Present 15B Present Absent Present Absent Present 16 Present Absent Present Absent Present 17 Present Absent Present Absent Present 18 Present Absent Present Absent Present


A2.2

Listeria Failure Rate


A B C D E Total


Total Results 16 16 16 16 16 80


= 1 / 16

= 6.3%


= 1 / 16

= 6.3%


= 0 / 16

= 0%


= 3 / 16

= 18.8%


= 1 / 16

= 6.3%

Overall failure rate (Listeria)


= 6 / 80

= 7.5%

A2.3

Listeria monocytogenes Results


Results 1 Present Absent Absent Absent Present 2 Present Absent Absent Absent Present 3 Present Absent Present Absent Present 1 4 Present Absent Absent Absent Present 5 Present Absent Absent Absent Present 6 Present Absent Absent Absent Present 7 Present Absent Absent Absent Present 9 Present Absent Absent Absent Present

11 Present Absent Absent Absent Present 12 Not done Not done Not done Not done Not done 13 Present Absent Absent Absent Present

15A Absent Not done Absent Not done Absent 2 15B Absent Not done Absent Not done Absent 2 16 Present Absent Absent Absent Present 17 Present Absent Absent Absent Present 18 Present Absent Absent Absent Present


A2.4

Listeria monocytogenes Failure Rate


A B C D E Total


Total Results 15 13 15 13 15 71


= 2 / 15

= 13.3%


= 0 / 13

= 0%


= 1 / 15

= 6.7%


= 0 / 13

= 0%


= 2 / 15

= 13.3%

Overall failure rate (L. monocytogenes)


= 5 / 71

= 7.0%

Section A3

Bacillus cereus

A3.1

Bacillus cereus Results


Results 1 Absent Present Absent Present Absent 2 Absent Present Absent Present Absent 4 Not done Present Absent Present Absent 6 Absent Present Absent Present Absent 7 Absent Present Absent Present Absent 9 Absent Present Absent Present Absent

11 Absent Present Absent Absent Absent 1 13 Absent Present Absent Present Absent

15A Absent Present Absent Present Absent 15B Absent Present Absent Present Absent 16 Absent Present Absent Present Absent 18 Absent Present Absent Present Absent


A3.2

Bacillus cereus Failure Rate


A B C D E Total


Total Results 11 12 12 12 12 59


= 0 / 11

= 0%


= 0 / 12

= 0%


= 0 / 12

= 0%


= 1 / 12

= 8.3%


= 0 / 12

= 0%

Overall failure rate (B. cereus)


= 1 / 59

= 1.7%

APPENDIX B

Summary of Methods

SECTION B1

Salmonella

Salmonella – Media Lab

Code Non-selective Selective 1 Selective 2 Detection/

Isolation 1 Detection/ Isolation 2 Confirmation

1 Buffered peptone water RVS broth MKTTn XLD BSA API 20E kit, O, Vi & H serotyping

2 Buffered peptone water MSC broth RV broth XLD BSA TSI, urea, L-lysine/LDC, β-galactosidase/ONPG, O

serotyping

3 Buffered peptone water RVS broth MKTTn XLD, BGA XLD, BGA Plate onto non-selective agar, Microbact 12 Vitek kit,

Oxoid ID kit

4.1 Buffered peptone water RVS broth, MKTTn M-broth Vidas SLM ELFA Not done Plate onto non-selective agar, API 10S kit, BAX

Salmonella PCR

4.2 Buffered peptone water MKTTn RVS broth XLD Hektoen Enteric

agar Plate onto non-selective agar, API 10S kit

5 Buffered peptone water SX-2 broth XLD Chromogenic

SMID2 Plate onto non-selective agar, L-lysine/LDC, β-galactosidase/ONPG, API 20E kit, O & Vi & H serotyping

6 Buffered peptone water RVS broth M-broth XLD

BSA, Salmonella Chromogenic agar

(Chrom SA)

Plate onto non-selective agar, Biomerieux API 20E kit, O, Vi & H serotyping

7 Buffered peptone water

MSC broth, RVS broth, M-broth RVS broth, MKTTn XLD, Tecra BSA Plate onto non-selective agar, TSI, Microbact 12A/E kit


ICS Strip – Immuno-

concentration (Vidas)

ICS-F broth Vidas SLM Not done Not done

9 Buffered peptone water MKTTn MSRV XLD BGS Plate onto non-selective agar, TSI, L-lysine/LDC, β-

galactosidase/ONPG, O serotyping

11 Buffered peptone water RVS broth, MKTTn RVS broth, MKTTn XLD, BGA (Brilliant

Green agar) XLD, BGA (Brilliant

Green agar) TSI, urea, L-lysine/LDC, β-galactosidase/ONPG, VP, indole, API system

12 Lactose broth RV (R10) M-broth Tecra VIA Salmonella Chromogenic agar API 20E kit

13 Buffered peptone water RVS broth MKTTn XLD BSA Glissuda, O, Vi & H serotyping, API 20E

14 Buffered peptone water MKTTn RVS broth XLD, MLCB agar SCA agar Plate onto non-selective agar, API 20E kit, O & H

serotyping

15A Buffered peptone water SX broth

XLD, Vidas, Chromogenic

media API 20E kit

15B Buffered peptone water SX broth

XLD, Vidas, Chromogenic

media API 20E kit

B1.1

Salmonella – Media Lab


Isolation 1 Detection/ Isolation 2 Confirmation

16 Buffered peptone water RVS broth, M-broth MKTTn XLD, Vidas BSA Plate onto non-selective agar, TSI, Microbact GNB 12A

kit, O, Vi & H serotyping


RVS broth, MKTTn, M-broth

RVS broth, MKTTn, M-broth XLD, BSA XLD, BSA

Plate onto non-selective agar, TSI, urea, L-lysine/LDC, β-galactosidase/ONPG, VP, indole, Microbact kit, O, Vi & H serotyping

18 Buffered peptone water RVS broth Not done Tecra Ultima XLD, Chromogenic

Salmonella agar Microbact 12E & Latex

B1.2

Salmonella – Incubation Temperature ( °°°°C) Salmonella – Duration of Incubation (hours) Lab


Isolation 1 Detection/ Isolation 2 Confirmation Non-

selective Selective 1 Selective 2 Detection/ Isolation 1

Detection/ Isolation 2 Confirmation

1 37 42 37 37 37 37 18 18 18 24 48 24

2 37 37 42 37 37 37 24 24 24 24 48 24

3 35 42 35 35 35 35 24 24 24 24 24 24

4.1 MKTTn: 37, RVS: 41.5 41.5 6 18

4.2 37 41.5 37 37 37 18 18 24 24 24

5 37 42 37 37 37 18 18 24 24 24

6 37 42 42 37 37 37 18 6 18 24 24, 48 24

7 37 37, 42 37, 42 37 37 37 18-22 6-8 6-8 24 48 24

8 37 41 24 5

9 37 37 42 37 37 37 24 24 24 24 24 24

11 37 37, 42 37, 42 37 37 37 24 24 24 24 24 24

12 37 42 37 37 37 18 24 8 24 24

13 37 42 37 37 37 37 24 24 24 24 24, 48 24

14 37 37 42 37 37 37 18 24 24 24 24 24

15A 37 42 37 24 24 24,48

15B 37 42 37 24 24 24,48

16 37 42 37 37 37 37 18 24 24 24 48 24

B1.3

Salmonella – Incubation Temperature ( °°°°C) Salmonella – Duration of Incubation (hours) Lab


Isolation 1 Detection/ Isolation 2 Confirmation Non-

selective Selective 1 Selective 2 Detection/ Isolation 1

Detection/ Isolation 2 Confirmation

17 37 37, 42 37, 42 37 37 37 18, 24 18, 24 18, 24 24, 48 24, 48 24

18 37 42 37 24 24 24, 48

B1.4

SECTION B2

Listeria

Listeria – Media Lab

Code Non-selective Primary Second

Selective Agar Plating 1 Agar Plating 2 Other Detection Confirmation

1 Not done Half Fraser (for BAX) MOPS BLEB Oxford agar Palcam agar BAX PCR DNA

probe β haemolysis, CAMP test, motility test, sugar fermentation tests

2 Not done Half Fraser broth

Full strength Fraser broth Oxford agar Palcam agar Not done Plate onto non-selective agar, β haemolysis, CAMP test,

motility test, sugar fermentation tests


Full strength Fraser broth Oxford agar Palcam agar Plate onto non-selective agar, motility test, API Listeria ID kit,

Gram stain, catalase

5 Half Fraser broth

Full strength Fraser broth OAA Vidas LIS

ELISA kit Plate onto non-selective agar, β haemolysis, motility test, API LIS ID kit


Full strength Fraser broth,

Listeria enrichment broth (LEB)

Oxford agar

Palcam agar, Chromocult Listeria agar (Chrom_LI)

Biomerieux VIDAS Listeria

ELISA kit, Dupont BAX DNA probe

Plate onto non-selective agar, CAMP test, Biomerieux API Listeria ID kit, Gram stain, catalase test


Full strength Fraser broth Oxford agar Palcam agar Not done Plate onto non-selective agar, β haemolysis, motility test,

Microbact 12L ID kit, Gram stain, catalase


Full strength Fraser broth Oxford agar Not done Plate onto non-selective agar, β haemolysis, CAMP test,

sugar fermentation tests, BAX PCR


Full strength Fraser broth ALOA Palcam agar API system

12 Tecra Listeria

enrichment broth

Full strength Fraser broth Tecra VIA

ELISA kit Not done

13 Half Fraser broth

Full strength Fraser broth Oxford agar Palcam agar BAX L. mono Listeria API ID kit, BAX L. mono

15A Not done Half Fraser broth

Full strength Fraser broth

Oxford agar, Sanoa Palcam agar Vidas Motility test, catalase, Gram stain

15B Not done Half Fraser broth

Full strength Fraser broth

Oxford agar, Sanoa Palcam agar Vidas Motility test, catalase, Gram stain


Full strength Fraser broth Oxford agar Palcam agar Vidas ELISA kit Plate onto non-selective agar, β haemolysis, motility test,

Microbact Listeria 12L ID kit

17 DFB FB FB Oxford agar, Palcam agar Palcam agar Microbact Microbact

18 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, motility test, Microbact 12L ID kit

B2.1

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.

1 30 35 37 37 37 24 24 48 48 24

2 30 37 37 37 37 24 48 48 48 48, sugars: up to 7 days

4 30 30 37 37 motility: 25 24 24 48 48 24

5 30 30 37 25, 37 24 24 48 24

6 30 30 30 37 37 24 24 48 48 24

7 30 37 37 37 25 24 48 48 48 48

9 30 37 37 37 24 48 48 24

11 30 35 37 37 37 24 48 24 24 24

12 30 30 48 24

13 30 37 37 37 37 24 48 48 48 24

15A 30 30 37 37 24 24 24, 48 48

15B 30 30 37 37 24 24 24, 48 48

16 30 30, 37 37 37 25, 37 24, 48 48 48 24, 48

17 30 30, 37 30, 37 37 37 37 37 24 24, 48 48 48 48 24 24

18 30 30 24 24

Note: Laboratory 3 did not provide any method information for Listeria.

B2.2

SECTION B3

Bacillus cereus

B. cereus – Media B. cereus – Plating / Inoculation Details

B. cereus – Incubation Temperature ( °°°°C)

B. cereus – Duration of Incubation (hours) Lab

Code Plating Method MPN Method Confirmation Plating Method MPN Method Plating Method MPN Method Plating Method MPN Method

1 PEMBA Not done Spore stain / Lipid stain 1 mL over 5 plates 37

24 / 24 on bench

(ambient)

2 BCSA Not done Spore stain / Lipid stain 0.1 mL per plate 37 24

4 MYP Not done Haemolysis (Sheep blood agar)

1 mL over 3 plates 30 48

6 MYP Not done Haemolysis (Sheep blood agar), Gram stain

1 mL over 3 plates 30, room temp 24

7 MYP Not done Haemolysis (Sheep blood agar), Spore stain / Lipid

stain, Gram stain


9 MYP Not done Haemolysis (Sheep blood agar)


11 MYP Not done API system (API CHB) 0.1 mL per plate 30 24

13 MYP Haemolysis (Sheep blood agar), Spore stain / Lipid

stain, Gram stain

1 mL over 3 plates, 0.1 mL

per plate 30 24, 48

15A PEMBA Not done Spore stain / Lipid stain 1 mL over 3 plates 37 24

15B PEMBA Not done Spore stain / Lipid stain 1 mL over 3 plates 37 24

16 MYP Not done Haemolysis (Sheep blood agar), Spore stain / Lipid

stain, Gram stain

1 mL over 3 plates 30 24, 48

18 Trypticase Soy Polymixin broth

Haemolysis (Sheep blood agar), Spore stain / Lipid

stain, MYP agar 10 mL of 10-1 30 48

B3.1

APPENDIX C

Homogeneity and

Stability Testing

C1.1

HOMOGENEITY TESTING

Ten samples from each matrix (A, B, C, D and E) were randomly chosen and tested by AsureQuality Limited (New Zealand) to confirm that the samples were homogeneous. Homogeneity testing was performed on 11 March 2009. 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: 2004 (ISO equivalent – ISO 6579: 2002). For Listeria, the method of testing was enumeration via spread plate technique using Palcam and Modified Oxford agars. The samples were verified using AS 5013.24.1: 2009 (ISO equivalent – ISO 11290-1: 1996, MOD). For Bacillus cereus, the method of testing was enumeration via spread plate technique using MYP agar. The samples were verified using AS 5013.2: 2007. The results are tabulated below.

Sample A (containing Listeria monocytogenes)

Sample Salmonella Listeria L. monocytogenes B. cereus 45 Not detected Detected Detected Not detected 48 Not detected Detected Detected Not detected 60 Not detected Detected Detected Not detected 65 Not detected Detected Detected Not detected 67 Not detected Detected Detected Not detected 81 Not detected Detected Detected Not detected 94 Not detected Detected Detected Not detected 164 Not detected Detected Detected Not detected 196 Not detected Detected Detected Not detected 202 Not detected Detected Detected Not detected

Sample B (containing Salmonella Adelaide and Bacillus cereus)

Sample Salmonella Listeria L. monocytogenes B. cereus 8 Detected Not detected Not detected Detected

26 Detected Not detected Not detected Detected 28 Detected Not detected Not detected Detected 44 Detected Not detected Not detected Detected 53 Detected Not detected Not detected Detected 58 Detected Not detected Not detected Detected 77 Detected Not detected Not detected Detected 94 Detected Not detected Not detected Detected 100 Detected Not detected Not detected Detected 111 Detected Not detected Not detected Detected

C1.2

Sample C (containing Salmonella Derby and Listeria innocua)

Sample Salmonella Listeria L. monocytogenes B. cereus 24 Detected Detected Not detected Not detected 30 Detected Detected Not detected Not detected 40 Detected Detected Not detected Not detected 51 Detected Detected Not detected Not detected 59 Detected Detected Not detected Not detected 67 Detected Detected Not detected Not detected 78 Detected Detected Not detected Not detected 89 Detected Detected Not detected Not detected 91 Detected Detected Not detected Not detected 108 Detected Detected Not detected Not detected

Sample D (containing Bacillus cereus)

Sample Salmonella Listeria L. monocytogenes B. cereus 4 Not detected Not detected Not detected Detected

17 Not detected Not detected Not detected Detected 19 Not detected Not detected Not detected Detected 21 Not detected Not detected Not detected Detected 28 Not detected Not detected Not detected Detected 52 Not detected Not detected Not detected Detected 70 Not detected Not detected Not detected Detected 73 Not detected Not detected Not detected Detected 102 Not detected Not detected Not detected Detected 116 Not detected Not detected Not detected Detected

Sample E (containing Salmonella Senftenberg and Listeria monocytogenes)

Sample Salmonella Listeria L. monocytogenes B. cereus 6 Detected Detected Detected Not detected 7 Detected Detected Detected Not detected

12 Detected Detected Detected Not detected 53 Detected Detected Detected Not detected 64 Detected Detected Detected Not detected 66 Detected Detected Detected Not detected 68 Detected Detected Detected Not detected 69 Detected Detected Detected Not detected 86 Detected Detected Detected Not detected 109 Detected Detected Detected Not detected

Based on the above testing results, the homogeneity of the samples was established.

C1.3

STABILITY TESTING 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 AsureQuality Limited (New Zealand). The stability testing took place on 23 April 2009. The results are tabulated below.

Sample A (containing Listeria monocytogenes)

Sample Salmonella Listeria L. monocytogenes B. cereus 49 Not detected Detected Detected Not detected 81 Not detected Detected Detected Not detected 113 Not detected Detected Detected Not detected

Sample B (containing Salmonella Adelaide and Bacillus cereus)

Sample Salmonella Listeria L. monocytogenes B. cereus 33 Detected Not detected Not detected Detected 95 Detected Not detected Not detected Detected 99 Detected Not detected Not detected Detected

Sample C (containing Salmonella Derby and Listeria innocua)

Sample Salmonella Listeria L. monocytogenes B. cereus 35 Detected Detected Not detected Not detected 49 Detected Detected Not detected Not detected 81 Detected Detected Not detected Not detected

Sample D (containing Bacillus cereus)

Sample Salmonella Listeria L. monocytogenes B. cereus 54 Not detected Not detected Not detected Detected 85 Not detected Not detected Not detected Detected 89 Not detected Not detected Not detected Detected

Sample E (containing Salmonella Senftenberg and Listeria monocytogenes)

Sample Salmonella Listeria L. monocytogenes B. cereus 40 Detected Detected Detected Not detected 67 Detected Detected Detected Not detected 84 Detected Detected Detected Not 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 19 April 2009 Page 1 of 5

PROFICIENCY TESTING AUSTRALIA PATHOGENS IN FOOD PROGRAM – ROUND 19

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 rice flour are to be tested for the presence /

absence of Salmonella and Listeria as per instructions below. Testing for the presence / absence of Bacillus cereus 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 B. cereus 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 rice flour 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 the freeze-dried matrix by adding 3.0 mL of sterile diluent (e.g. 0.1% (w/v) peptone and 0.85%

(w/v) NaCl (ISO, 1988)) 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 rice flour. Add 1 mL of the rehydrated vial contents and continue as per your Standard method. Please note: B. cereus testing: Aliquots are to be removed from the initial non-selective Salmonella enrichment once prepared. If following the plating procedure for B. cereus, levels have been designed to suit the inoculum volume of 1 mL (over 3 plates) if following AS 5013. 2 – 2007.

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 B. cereus 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 5 May 2009 to: Mark Bunt Proficiency Testing Australia PO Box 7507 Silverwater NSW 2128 Telephone: +61 2 9736 8397 (1300 782 867) AUSTRALIA Fax: +61 2 9743 6664


PTA Pathogens in Food (Round 19) 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

Bacillus cereus � Present

� Absent

� Not done

� Present

� Absent

� Not done

� Present

� Absent

� Not done

� Present

� Absent

� Not done

� Present

� Absent

� Not done

D2.1


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

� 35ºC � 37ºC � 42ºC � Other ____ ºC

� 35ºC � 37ºC � 42ºC � Other ____ ºC

� 35ºC � 37ºC � 42ºC � Other ____ ºC

� 35ºC � 37ºC � 42ºC � Other _____ ºC

� 35ºC � 37ºC � 42ºC � Other _____ ºC

� 35ºC � 37ºC � 42ºC � 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 __________

D

2.2


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 � Other agar (specify) _____________

� Not done � Palcam agar � 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

� 25ºC � 30ºC � 37ºC � Other _____ ºC

� 25ºC � 30ºC � 37ºC � Other _____ ºC

� 25ºC � 30ºC � 37ºC � Other _____ ºC

� 25ºC � 30ºC � 37ºC � Other _____ ºC

� 25ºC � 30ºC � 37ºC � Other _____ ºC

� 25ºC � 30ºC � 37ºC � Other _____ ºC

� 25ºC � 30ºC � 37ºC � Other _____ ºC

Duration of Incubation

� 4 hours � 24 hours � 48 hours � Other __________







D2.3


Table D: Method Information: Bacillus cereus (Presence/Absence) Laboratory Code Please indicate the methodology used by filling in (•) in the appropriate circles below:

Method Plating Method Most Probable Number Method C onfirmation

Media

� MYP

� KG

� Other (specify)

_______________________

� Not done

� Trypticase Soy Polymixin broth

� Other (specify)

_______________________

Plating / Inoculation Details

� 1 mL over 3 plates (recommended) � 0.1 mL per plate � Other (specify)

____________________

� 0.1 g (1 mL of 10-1) � 0.01 g (1 mL of 10-2) � 0.001 g (1 mL of 10-3) � Other (specify)

_____________________

Incubation Temperature � 30 ºC

� Other ______ ºC � 30 ºC � Other ______ ºC

Duration of Incubation � 18 hours

� 24 hours � 48 hours � Other ____________

� 48 hours � Other ____________

� Not done

� Haemolysis (Sheep blood agar)

� Spore stain / Lipid stain

� Gram stain

� Motility

� Nitrate reduction

� Voges Proskauer (VP)

� Anaerobic glucose fermentation

� Tyrosine decomposition

� Toxin crystals stain

� Lysozyme resistance

� Other (specify)

_______________________________

D

2.4

----- End of report -----