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Prepared on behalf of FEPAS® by

Rosemary A Smith

© Crown Copyright 2006 of 36 0009 PT

FEPAS® Assessment 109

February 2006

Report

ELECTRONIC COPY Only participants can assess their performance in this proficiency test by obtaining their laboratory number from FAPAS SecureWeb or direct from FAPAS®.

FEPAS® Assessment 109 ELECTRONIC COPY

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SUMMARY

1. The test materials for FEPAS® Assessment 109 were dispatched in February 2006. Test materials for seven examinations were available in Assessment 109: enumeration of coagulase positive staphylococci (10 g equivalent, chicken) M0837, enumeration of Enterobacteriaceae (10 g, milk powder) M0317, aerobic plate count (10 g, milk powder) M0947, enumeration of lactic acid bacteria (10 g equivalent, beef) M0118, detection of Salmonella spp. (25 g, chicken) M0758, detection of Listeria monocytogenes (25 g, milk powder) M0238 and detection of E. coli O157 (25 g equivalent, beef) M1030. Of the 162 participants to receive test materials, 154 participants, i.e. 95%, returned results for one or more examinations within the time-scale demanded by the Scheme.

2. The assigned value ( X̂ ) for each enumeration examination was calculated from the most appropriate measure of central tendency of participants’ results [1, 2, 3]. Qualitative results were assessed based on the presence or absence of the target organism in the test materials issued.

3. The target standard deviation (σp) was calculated using retrospective FEPAS® data and in

conjunction with the assigned value ( X̂ ) was used to derive a z-score for participants’ results. z-Scores are considered satisfactory if |z| ≤2.

4. Results for this Assessment are summarized as follows:

test assigned value (log10cfu/g)

/ assessment

n data points

used for assigned value

number of satisfactory z-scores

or assessments

total number of results

satisfactory%

enumeration of coagulase positive

staphylococci

4.15 52 47 53 89

enumeration of Enterobacteriaceae

3.74 32 28 33 85

aerobic plate count 4.56 34 31 34 91

enumeration of lactic acid bacteria

3.15 16 16 16 100

detection of Salmonella spp.

TM A present TM B present

n/a 31 34 91

detection of L. monocytogenes

TM A present TM B absent

n/a 47 51 92

detection of E. coli O157

TM A present TM B absent

n/a 17 22 77

n/a = not applicable


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5. Whereas this report has been produced in good faith and in accordance with best industry practice, neither the Central Science Laboratory nor the Secretary of State for Environment, Food and Rural Affairs accepts any liability whatsoever as to the application or use of the information contained therein.


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CONTENTS 1. INTRODUCTION 5

1.1 Proficiency Testing 5 1.2 Spoilage Organisms, Hygiene Indicators and Pathogens 5

2. TEST MATERIALS 6 2.1 Homogeneity 6 2.2 Distribution 6

3. RESULTS 7 4. STATISTICAL EVALUATION OF RESULTS 7

4.1 Calculation of the Assigned Value, X̂ 7 4.2 Target Standard Deviation for the Tests, σp 8 4.3 Individual z-Scores 8

5. REFERENCES 9 TABLES Table 1: Results and z-Scores for Coagulase Positive Staphylococci (Chicken) 10 Table 2: Results and z-Scores for Enterobacteriaceae (Milk Powder) 12 Table 3: Results and z-Scores for Aerobic Plate Count (Milk Powder) 14 Table 4: Results and z-Scores for Lactic Acid Bacteria (Beef) 16 Table 5: Results and Assessments for Salmonella spp. (Soybean Meal) 17 Table 6: Results and Assessments for L. monocytogenes (Milk Powder) 19 Table 7: Results and Assessments for E. Coli O157 (Beef) 21 Table 8: Assigned Values and Target Standard Deviations 22 Table 9: Numbers and Percentages of Satisfactory z-Scores and Assessments 22 FIGURES Figure 1: z-Scores for Coagulase Positive Staphylococci (4.15 log10cfu/g) in Chicken

Test Material 23 Figure 2: z-Scores for Enterobacteriaceae (3.74 log10cfu/g) in Milk Powder

Test Material 24 Figure 3: z-Scores for Aerobic Plate Count (4.56 log10cfu/g) in Milk Powder

Test Material 25 Figure 4: z-Scores for Lactic Acid Bacteria (3.15 log10cfu/g) in Beef Test Material 26 APPENDICES APPENDIX I: Homogeneity Data for Test Materials (Fearn and Thompson Test) 27

APPENDIX II: Homogeneity Data for Test Materials (F-Test) 28

APPENDIX III: Verification of Positive Qualitative Test Materials 29

APPENDIX IV: Organisms present in FEPAS® Test Materials 29

APPENDIX V: Microbiological Methods Used by Participants 30

APPENDIX VI: Reports and Protocol 36


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1. INTRODUCTION

1.1 Proficiency Testing

The demand for independent proof of competence from regulatory bodies and customers means that proficiency testing is relevant to all laboratories testing food and feed for quality and safety in every country. Hence, it is a requirement of accreditation to ISO 17025 [4] that the laboratory takes part in a proficiency testing scheme, if a suitable scheme exists. Further, for laboratories entrusted with the official control of food and feeds, Article 12 of EU Regulation (EC) 882/2004 [5] requires such laboratories to be assessed and accredited in accordance with ISO 17025, i.e. proficiency testing is a legal requirement for these laboratories. Thus, together with the use of validated methods, proficiency testing is an essential element of laboratory quality assurance.

The analysis of an external quality check sample as part of a laboratory’s routine procedures provides objective standards for individual laboratories to perform against and permits them to compare their microbiological results with those from other laboratories. Such standards and comparisons can go beyond the actual microbiological examination. For example, the ability to report results in specified units and within a given time scale are important aspects of quality. Hence, participants in FEPAS® who submit results after the closing date of a test are only included in the statistical evaluation if there are extenuating circumstances.

It is important to understand the statistical limitations of this external means of quality assessment when gauging the competence of a laboratory. When converted to logarithms the results of a typical microbiological examination will be normally distributed. That is to say, the majority of results will be centred on a mean value but, inevitably, some results will lie at the extremes of the distribution. The statistics of a normal distribution mean that about 95% of data points will lie between a z-score of –2 and +2. Performance in a FEPAS® proficiency test, therefore, is considered ‘satisfactory’ if a participant’s z-score lies within this range. It follows that if a participant’s z-score lies outside |z| >2 there is about a 1 in 20 chance that their result is in fact an acceptable result from the extreme of the distribution. If a participant’s z-score lies outside |z| >3 the chance that their result is actually acceptable is only about 1 in 300.

1.2 Spoilage Organisms, Hygiene Indicators and Pathogens

Enterobacteriaceae are useful indicators of hygiene and of post process contamination of heat-treated foods. Coliforms are members of the Enterobacteriaceae family that ferment lactose and include Enterobacter, most Escherichia, Klebsiella and Citrobacter [6]. Methods are usually based on direct enumeration using media containing bile salts and lactose.

Enumeration of microorganisms by total viable count (aerobic plate count) is one of the most common methods used to indicate microbiological quality of a food. The temperature and length of incubation should be defined. The most common regime is 30oC for 48 or 72 hours for mesophilic organisms [6].

Microbiological spoilage of food can be defined as the result of metabolic activities of one or more microbial groups leading to adverse sensory changes in the specific product [7]. Lactic acid bacteria commonly found in food include Lactobacillus, Leuconostoc and Pediococcus. Starter cultures for fermented products such as cheese and salami commonly include this group of organisms and so their presence in high numbers should be expected. However, in


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some foods they may cause spoilage. Lactic acid bacteria show similar physiological behaviour on a number of elective and selective media with de Man, Rogosa and Sharpe (MRS) agar being the media of choice.

Salmonella spp. are organisms of faecal origin, and the majority of serovars are regarded as potentially pathogenic to humans. Methods must be capable of detecting low levels of this pathogen in the presence of high levels of background flora [8]. Traditional procedures are lengthy and include a pre-enrichment in a non-selective medium, enrichment in selective media and subculture to solid selective media.

L. monocytogenes is ubiquitous in the environment. Large outbreaks of listeriosis have been linked to raw cabbage, soft cheese, pâté and pork tongues [9] and more recently butter and sandwiches [10]. As the organism is able to grow at refrigeration temperatures, methods must be capable of detecting low levels of this pathogen in the presence of high levels of background flora [9]. Traditional procedures are lengthy and include enrichment in selective media and subculture to solid selective media.

Whilst the presence of E. coli in food is generally undesirable because it indicates poor hygienic conditions, certain serotypes are pathogenic. Verocytotoxin producing strains of E. coli (VTEC) e.g. E. coli O157 may be particularly virulent. Selective media currently used for the isolation of E. coli O157 are based on the strain's inability to ferment sorbitol [6].

2. TEST MATERIALS

2.1 Homogeneity

It has been proposed that the statistical test for homogeneity previously used by FEPAS® should be replaced by a new test [11] in order to overcome the frequently encountered problem of rejection of material which is in fact ‘fit for purpose’. In this report, both the previous test and the new test for homogeneity are presented in APPENDICES I and II for comparison.

The revised test [11] initially checks the data for any widely discrepant pairs using Cochran’s test. If found, such data are removed. Thereafter, the remaining data are subject to analysis of variance (ANOVA) to estimate the sampling and analytical variances. The ‘F’ test presents the total colony forming units (cfu) across duplicate plates whereas this test requires cfu/g to be transformed into log10cfu/g.

The results, together with their statistical evaluations, are given in APPENDICES I and II. These data show sufficient homogeneity, and are not included in the subsequent calculations of the assigned values.

Verification data for the detection examinations are presented in APPENDIX III.

FEPAS® test materials include background flora to simulate real conditions. Information on these organisms is given in APPENDIX IV.

2.2 Distribution

Each participant received one or more individually numbered FEPAS® test material vials together with a covering letter, instructions for the electronic submission of results and


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methods and a results form for those participants with no access to the Internet. The dispatch date was 06 February 2006.

3. RESULTS

Participants were required to report their data in either cfu/g or detected / not detected in 25 g as appropriate. Results were submitted by 154 participants before the closing date for this Assessment, 24 February 2006.

Each participant was given a laboratory number, assigned in order of receipt of results. The reported results are given in Table 1-Table 7.

The microbiological methods used by each participant are summarised in APPENDIX V.

4. STATISTICAL EVALUATION OF RESULTS

The object of the statistical procedure employed is to obtain a simple and transparent result, which the participant and other interested parties can readily appreciate. Further details, including worked examples, are given in the FEPAS® Protocol [2]. The procedure follows that recommended in the IUPAC/ISO/AOAC International Harmonised Protocol for the Proficiency Testing of (Chemical) Analytical Laboratories [12].

4.1 Calculation of the Assigned Value, X̂

The assigned value, X̂ , i.e. the best estimate of the true number of each organism, was set as the consensus of the results submitted by participants. The procedure used to derive this consensus involved:

• Log transforming participant results from cfu/g to log10cfu/g. • Removal of non-valid data • Using a robust statistical procedure to derive the mean [1] of the final data set. • Assessing the uncertainty of the robust mean. • Considering the normality (Kolmogorov-Smirnov test), or otherwise, of the distribution

of the selected results.

• Scrutinising the data, if appropriate, with a procedure to identify multi-modality (bump-hunting) [3].

For the enumeration of coagulase positive staphylococci and Enterobacteriaceae, the results submitted by laboratory 074 were not included for the calculation of the robust mean because the laboratory did not receive the samples until after the deadline for examination.

For the enumeration of coagulase positive staphylococci, the enumeration of Enterobacteriaceae, the aerobic plate count and the enumeration of lactic acid bacteria, the procedure for the derivation of the concensus from the remaining valid data was straightforward. The robust mean was shown to be the best measure of central tendency and was therefore set as the assigned value [1] for each enumeration. The assigned values, X̂ , are shown in Table 8.


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4.2 Target Standard Deviation for the Tests, σp

The value of σp is set at a value which reflects ‘best practice’ for the analysis in question. σp has therefore been estimated from previous FEPAS® data and the FEPAS® Advisory Committee has agreed that an appropriate value is 0.25. This value for σp has been used to calculate z-scores from the reported results for the enumeration exercises in this assessment.

4.3 Individual z-Scores

Participants’ z-scores were calculated as:

p

Xxzσ

)ˆ( −=

where x is the reported value for the number of microorganisms (converted to logarithms) in the test material,

X̂ is the assigned value, the best estimate of the “true” number of microorganisms (converted to logarithms),

and σp is the target standard deviation.

Participants’ results and assessments for each enumeration examination are given in Table 1-Table 4 and shown as histograms in Figure 1-Figure 4.

Qualitative results are assessed based on the presence or absence of the target organism in the test materials issued. Assessments for each of the detection examinations are given in Table 5-Table 7.

The numbers and percentages of z-scores in the satisfactory range, |z| ≤2, and satisfactory assessments for each examination are given in Table 9.


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5. REFERENCES 1 Analytical Methods Committee, 1989, Robust Statistics – How not to reject outliers

Part 1. Basic Concepts, Analyst, 114, 1693-1697.

2 FEPAS®, 2002, Protocol for the Organisation and Analysis of Data, 3rd Edition.

3 Lowthian, P.J. and Thompson, M., 2002, Bump-hunting for the proficiency tester – searching for multimodality, Analyst, 127, 1359-1364.

4 ISO/IEC 17025:2005, General requirements for the competence of testing and calibration laboratories.

5 Regulation (EC) 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules, Official Journal L 165, 30/04/2004, 0001-0141.

6 Bell, C., Greenwood, M., Hooker, J., Kyriakides, A. & Mills, R., 1999, Development and Use of Microbiological Criteria for Foods, Institute of Food Science and Technology (UK), London

7 Holzapfel, W.H., 1995, Culture media for non-sporulating Gram-positive food spoilage bacteria. In: Culture Media for Food Microbiology. Ed: Corry, J.E.L., Curtis, G.D.W. & Baird, R.M., Elsevier Science, Amsterdam, The Netherlands.

8 Busse, M., 1995, Media for Salmonella. In: Culture Media for Food Microbiology. Ed Corry, J.E.L., Curtis, G.D.W & Baird, R.M., Elsevier Science, Amsterdam, The Netherlands.

9 Curtis, G.D.W. & Lee, W.H., 1995, Culture media and methods for the isolation of Listeria monocytogenes. In: Culture Media for Food Microbiology. Ed Corry, J.E.L., Curtis, G.D.W. & Baird, R.M., Elsevier Science, Amsterdam, The Netherlands.

10 Advisory Committee on the Microbiological Safety of Food (ACMSF), 2003, Information Paper, Recent trends in Listeriosis in the UK. ACM/667

11 Fearn, T. and Thompson, M., 2002, A new test for ‘sufficient homogeneity’. Analyst, 126, 1414-1417.

12 Thompson, M. and Wood, R., 1993, The International Harmonised Protocol for the Proficiency Testing of (Chemical) Analytical Laboratories, Journal of the Association of Official Analytical Chemists International, 76(4), 929-940.


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Table 1: Results and z-Scores for Coagulase Positive Staphylococci (Chicken)

laboratory number coagulase positive staphylococci

assigned value 4.15 log10cfu/g

result, cfu/g result, log10cfu z-score

001 1.6E+4 4.204 0.2

008 2.0E+4 4.301 0.6

009 1.6E+4 4.204 0.2

010 2.42E+4 4.384 0.9

013 1.7E+3 3.230 -3.7

020 2.1E+4 4.322 0.7

023 1.0E+4 4.000 -0.6

027 5.2E+3 3.716 -1.7

028 1.8E+4 4.255 0.4

031 2.07E+4 4.316 0.7

032 1.6E+4 4.204 0.2

034 1.9E+4 4.279 0.5

035 1.8E+4 4.255 0.4

037 7.5E+3 3.875 -1.1

038 2.1E+4 4.322 0.7

043 1.25E+4 4.097 -0.2

047 9.4E+3 3.973 -0.7

048 2.24E+4 4.350 0.8

050 1.7E+4 4.230 0.3

051 2.4E+4 4.380 0.9

052 5.3E+3 3.724 -1.7

053 1.7E+4 4.230 0.3

054 Negative

060 1.64E+4 4.215 0.3

064 1.2E+4 4.079 -0.3

065 11000 4.041 -0.4

070 1.5E+5 5.176 4.1

071 2.1E+4 4.322 0.7

Results are presented as reported by participants using the ESR system z-Scores outside the satisfactory range are shown in bold


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Table 1(continued): Results and z-Scores for Coagulase Positive Staphylococci (Chicken)

laboratory number coagulase positive staphylococci



073 1.3E+4 4.114 -0.1

074 * 6.3E+3 3.799 -1.4

078 1.3E+4 4.114 -0.1

080 7.2E+3 3.857 -1.2

081 1.2E+5 5.079 3.7

092 7700 3.886 -1.0

094 * 4.5E+3 3.653 -2.0

095 14000 4.146 0.0

097 22000 4.342 0.8

101 1.7E+4 4.230 0.3

102 2E+4 4.301 0.6

103 1.8E+3 3.255 -3.6

109 8.0E+3 3.903 -1.0

113 8.8E+3 3.944 -0.8

117 1.5E4 4.176 0.1

125 2.1E+4 4.322 0.7

127 11500 4.061 -0.3

130 37000 4.568 1.7

137 2.0E+3 3.301 -3.4

140 21300 4.328 0.7

147 3.2E+4 4.505 1.4

148 18100 4.258 0.4

149 1.8E+4 4.255 0.4

150 4.2E+3 3.623 -2.1

151 7000 3.845 -1.2

152 2.3E+4 4.362 0.9

Results are presented as reported by participants using the ESR system z-Scores outside the satisfactory range are shown in bold * Sample tested outside of recommended time period


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Table 2: Results and z-Scores for Enterobacteriaceae (Milk Powder)

laboratory number Enterobacteriaceae



003 3.75E+3 3.574 -0.7

014 3.2E+3 3.505 -0.9

015 4.6E+3 3.663 -0.3

018 8.6E+3 3.934 0.8

021 4.0E+3 3.602 -0.6

029 2.2E+4 4.342 2.4

031 4.46E+3 3.649 -0.4

032 1.7E+4 4.230 2.0

051 7.2E+3 3.857 0.5

065 12800 4.107 1.5

066 3.0E+3 3.477 -1.1

069 3.5E+3 3.544 -0.8

074 1.8E+3 3.255 -1.9

075 5E+4 4.699 3.8

078 1.1E+4 4.041 1.2

083 7.7E+3 3.886 0.6

084 4.4E+3 3.643 -0.4

091 7636.4 3.883 0.6

092 3400 3.531 -0.8

093 6.4E+3 3.806 0.3

094 2.0E+3 3.301 -1.8

098 7.1E+3 3.851 0.4

103 4.7E+3 3.672 -0.3

109 3.0E+3 3.477 -1.1

110 1.3E+3 3.114 -2.5

116 240 2.380 -5.4

117 7.6E+3 3.881 0.6

125 4.4E+3 3.643 -0.4



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Table 2 (continued): Results and z-Scores for Enterobacteriaceae (Milk Powder)

laboratory number Enterobacteriaceae



130 5766 3.761 0.1

133 8E+3 3.903 0.6

141 4.0E+4 4.602 3.4

150 2.7E+3 3.431 -1.2

151 5.5E+3 3.740 0.0



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Table 3: Results and z-Scores for Aerobic Plate Count (Milk Powder)

laboratory number aerobic plate count



002 4.5E+4 4.653 0.4

004 5.6E+4 4.748 0.7

007 4.7E+4 4.672 0.4

014 26E+3 4.415 -0.6

016 2.4E+4 4.380 -0.7

017 4.8E+4 4.681 0.5

021 2.8E+4 4.447 -0.5

022 2300 3.362 -4.8

024 4220 3.625 -3.8

025 3.8E+4 4.580 0.1

026 4.2E+4 4.623 0.2

032 6E+4 4.778 0.9

033 3.4E+4 4.531 -0.1

041 40000 4.602 0.1

042 2.3E+4 4.362 -0.8

046 2.9575E+4 4.471 -0.4

048 4.31E+4 4.634 0.3

051 4.1E+4 4.613 0.2

058 5.2E+4 4.716 0.6

069 1.2E+4 4.079 -1.9

085 3.4E+4 4.531 -0.1

086 3.1E+4 4.491 -0.3

094 * 2.0E+4 4.301 -1.1

103 5.6E+4 4.748 0.7

111 4.6E+4 4.663 0.4

118 4.2E+4 4.623 0.2

122 3.4e+4 4.531 -0.1

124 3.0E+4 4.477 -0.4

Results are presented as reported by participants using the ESR system * Sample tested outside of recommended time period z-Scores outside the satisfactory range are shown in bold


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Table 3 (continued): Results and z-Scores for Aerobic Plate Count (Milk Powder)

laboratory number aerobic plate count



125 4.9E+4 4.690 0.5

129 5.1E+4 4.708 0.6

131 4.6E+4 4.663 0.4

150 3.2E+5 5.505 3.8

151 3.0E+4 4.477 -0.4

152 4.0E+4 4.602 0.1



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Table 4: Results and z-Scores for Lactic Acid Bacteria (Beef)

laboratory number lactic acid bacteria



005 1.8E3 3.255 0.4

019 1.8E+3 3.255 0.4

028 1.1E+3 3.041 -0.4

039 1.2E+3 3.079 -0.3

045 1.4E+3 3.146 0.0

065 1250 3.097 -0.2

078 1.4E+3 3.146 0.0

087 1.6E+3 3.204 0.2

096 6.0E+2 2.778 -1.5

097 2300 3.362 0.8

104 1.53E+3 3.185 0.1

105 7.9e+2 2.898 -1.0

108 1.8E3 3.255 0.4

127 1780 3.250 0.4

141 3.9E+3 3.591 1.8

153 930 2.968 -0.7

Results are presented as reported by participants using the ESR system


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Table 5: Results and Assessments for Salmonella spp. (Soybean Meal)

laboratory number Salmonella spp.

test material A test material B

result result satisfactory /

PRESENT PRESENT not satisfactory

006 detected detected S




024 not detected not detected NS




049 detected not detected NS


056 * detected detected S







075 * detected detected S











Results are presented as reported by participants using the ESR system * Sample tested outside of recommended time period S = Satisfactory NS = Not Satisfactory


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Table 5 (continued): Results and Assessments for Salmonella spp. (Soybean Meal)

laboratory number Salmonella spp.



PRESENT PRESENT not satisfactory




135 * not detected detected NS





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Table 6: Results and Assessments for L. monocytogenes (Milk Powder)

laboratory number L. monocytogenes



PRESENT ABSENT not satisfactory

009 detected not detected S



056 * detected not detected S





066 detected detected NS






















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Table 6 (continued): Results and Assessments for L. monocytogenes (Milk Powder)

laboratory number L. monocytogenes





















142 not detected detected NS






Results are presented as reported by participants using the ESR system S = Satisfactory NS = Not Satisfactory


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Table 7: Results and Assessments for E. coli O157 (Beef)

laboratory number E. coli O157


result result satisfactory/













094 * detected detected NS








136 * detected detected NS





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Table 8: Assigned Values and Target Standard Deviations

test X̂ robust mean

log10cfu/g

or present/absent

n data points

σp from retrospective

FEPAS® data

coagulase positive staphylococci 4.15 52 0.25

Enterobacteriaceae 3.74 32 0.25

aerobic plate count 4.56 34 0.25

lactic acid bacteria 3.15 16 0.25

Salmonella spp. TM A present TM B present

n/a n/a

L. monocytogenes TM A present TM B absent

n/a n/a

E. coli O157 TM A present TM B absent

n/a n/a


Table 9: Numbers and Percentages of Satisfactory z-Scores and Assessments

test number of satisfactory scores|z| ≤2 and assessments

total number

of results

satisfactory%

coagulase positive staphylococci 47 53 89

Enterobacteriaceae 28 33 85

aerobic plate count 31 34 91

lactic acid bacteria 16 16 100

Salmonella spp. 31 34 91

L. monocytogenes 47 51 92

E. coli O157 17 22 77


13 10313715094 27 52 74 15180 37 92 10911347 23 65 12764 43 73 78 95 1171 9 32 60 50 53 10128 35 14914834 8 10231 20 38 71 12514097 48 15251 10 14713081 70

3.65 log10cfu/g

4.15 log10cfu/g

4.65 log10cfu/g

-4.0

-3.0

-2.0

-1.0

0.0

1.0

2.0

3.0

4.0

Laboratory Number

z-score

Figure 1: z-Scores for Coagulase Positive Staphylococci (4.15 log10cfu/g) in Chicken Test Material

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116

110

74 94 150

66 109

14 92 69 3 21 84 125

31 15 103

151

130

93 98 51 117

91 83 133

18 78 65 32 29 141

75

4.24 log10cfu/g

3.74 log10cfu/g

3.24 log10cfu/g

-4.0

-3.0

-2.0

-1.0

0.0

1.0

2.0

3.0

4.0

Laboratory Number

z-score

Figure 2: z-Scores for Enterobacteriaceae (3.74 log10cfu/g) in Milk Powder Test Material

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22 24 69 94 42 16 14 21 46 124

151

86 33 85 122

25 41 152

51 26 118

48 2 111

131

7 17 125

129

58 4 103

32 150

5.06 log10cfu/g

4.56 log10cfu/g

4.06 log10cfu/g

-4.0

-3.0

-2.0

-1.0

0.0

1.0

2.0

3.0

4.0

Laboratory Number

z-score

Figure 3: z-Scores for Aerobic Plate Count (4.56 log10cfu/g) in Milk Powder Test Material

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96 105

153

28 39 65 45 78 104

87 127

5 19 108

97 141

3.65 log10cfu/g

3.15 log10cfu/g

2.65 log10cfu/g

-3.0

-2.0

-1.0

0.0

1.0

2.0

3.0

Laboratory Number

z-score

Figure 4: z-Scores for Lactic Acid Bacteria (3.15 log10cfu/g) in Beef Test Material

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APPENDIX I: Homogeneity Data for Test Materials (Fearn and Thompson Test)

sample identity organism (log10cfu/g)

coagulase positive staphylococci

Enterobacteriaceae aerobic plate count lactic acid bacteria

replicate 1 replicate 2 replicate 1 replicate 2 replicate 1 replicate 2 replicate 1 replicate 2

1 4.54 4.60 3.85 3.75 4.57 4.54 3.28 3.40

2 4.64 4.60 3.76 3.89 4.46 4.58 3.46 3.28

3 4.65 4.66 3.86 3.82 4.49 4.61 3.34 3.30

4 4.64 4.71 3.87 3.83 4.54 4.59 3.26 3.20

5 4.67 4.70 3.66 3.78 4.59 4.60 3.20 3.28

6 4.69 4.64 3.76 3.70 4.51 4.49 3.11 3.32

7 4.71 4.65 3.85 3.87 4.43 4.59 3.23 3.18

8 4.67 4.60 3.82 3.88 4.48 4.48 3.26 3.26

9 4.71 4.67 3.75 3.64 4.54 4.58 3.28 3.28

10 4.69 4.75 3.70 3.73 4.48 4.59 3.30 3.11

mean 4.66 3.79 4.54 3.27

n 20 20 20 20

abs. target sd (σp) 0.250 0.250 0.250 0.250

san 0.037 0.056 0.060 0.084

sam2 0.001 0.003 0.000 0.000

σall2 0.006 0.006 0.006 0.006

critical 0.012 0.014 0.014 0.018

sam2<critical? ACCEPT ACCEPT ACCEPT ACCEPT

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APPENDIX II: Homogeneity Data for Test Materials (F-Test)

sample identity organism (cfu/plate)


-3 dilution

Enterobacteriaceae -4 diliution

aerobic plate count -2 dilution

lactic acid bacteria -2 dilution

replicate 1 replicate 2 replicate 1 replicate 2 replicate 1 replicate 2 replicate 1 replicate 2

1 6 16 35 28 76 72 35 50

2 5 10 29 39 55 77 61 41

3 11 4 36 33 60 83 46 40

4 8 6 37 34 70 77 35 34

5 5 8 23 30 76 79 34 40

6 3 6 29 25 67 67 26 42

7 6 14 35 37 51 75 36 31

8 13 8 33 38 62 61 33 37

9 7 5 28 22 70 78 38 33

10 7 14 25 27 59 79 41 25

mean 8.1 31.2 69.7 37.9

n 20 20 20 20

F 0.66 2.72 0.53 0.94

F critical 3.02 3.02 3.02 3.02

F<Fcrit? PASS PASS PASS PASS

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APPENDIX III: Verification of Positive Qualitative Test Materials

target organism level of target organism (cfu/g)

Salmonella spp. 50

L. monocytogenes 43 - 93

E. coli O157 2.3 - 4.3

APPENDIX IV: Organisms present in FEPAS® Test Materials

test matrix target organism background flora


chicken Staphylococcus aureus Saccharomyces cerevisiae Pseudomonas putida Micrococcus luteus

Enterobacteriaceae milk powder E. coli Pseudomonas aeruginosa Enterococcus faecalis

aerobic plate count milk powder Bacillus cereus Lactobacillus plantarum

Ps aeruginosa

n/a

lactic acid bacteria beef L. plantarum Lactococcus lactis

M. luteus Ps. putida

S. cerevisiae

Salmonella spp. (all samples were

positive)

soybean meal Salmonella Poona natural background flora

L. monocytogenes (positive sample)

milk powder L. monocytogenes B. cereus S. aureus

L. plantarum

L. monocytogenes (negative sample)

milk powder Listeria ivanovii B.cereus S. aureus

L. plantarum

E. coli O157 (positive sample)

beef E.coli O157 Ps. putida M. luteus

E. coli

E. coli O157 (negative sample)

beef n/a Ps. putida M. luteus

E. coli



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APPENDIX V: Microbiological Methods Used by Participants

Notes

1) Participants with performance |z| >2 are no longer shown in bold within this Appendix. Please refer to Tables 1-7 for this information.

2) Participants’ methods are tabulated according to the information submitted electronically, but some responses may have been combined or edited for clarity.

Coagulase Positive Staphylococci

Accredited Method Used laboratory number

yes 001 008 013 020 031 032 034 035 037 038 043 047 051 052 053 064 073 078 080 081 094 095 101 102 109 113 117 125 127 137 140

no 010 050 060 065 070 071 074

Coagulase Positive Staphylococci Media laboratory number

Baird Parker Agar 008 009 013 020 023 028 031 032 034 035 038 047 050 051 053 060 064 070 071 074 081 094 102 109 117 125 127 137 140

Baird Parker Agar + RPF 001 010 034 073 078 095 113 130

Petrifilm 027 037 043 052 065 080

Petrifilm Staph Express 35 C, 24 H. 101

Salt Blood Agar 048

Enterobacteriaceae


yes 021 029 031 032 051 083 091 093 094 109 117 125 141

no 014 015 018 065 066 069 075 084

Enterobacteriacae Media laboratory number

Petrifilm 069

Violet Red Bile Agar 130 133

Violet Red Bile Glucose Agar 003 014 015 018 021 029 031 032 051 065 066 074 075 078 083 084 091 093 094 098 109 117 125 133 141


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Aerobic Plate Count


yes 007 014 021 024 032 048 051 086 094 118 122 124 129

no 002 004 016 022 025 033 042 046 058 069 085 125

Aerobic Plate Count Media laboratory number

Milk Plate Count Agar 017 032 086

Plate Count Agar 002 004 007 014 016 021 022 025 026 033 041 046 048 051 085 094 118 122 124 125

Petrifilm 024 042 058 069 129 131

Lactic Acid Bacteria


yes 019 028 039 045 078 104 141

no 005 065 087 096 105 108 127

Lactic Acid Bacteria Media laboratory number

de Man, Rogosa and Sharpe (MRS) Agar 005 028 039 045 065 078 087 096 104 105 108 127 141


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Salmonella spp.


yes 007 011 021 032 044 047 049 055 057 059 064 078 086 093 094 099 100 115 121 122 124 135

no 006 056 058 062 069 075(tmA) 088

Salmonella spp. Enrichment laboratory number

Buffered Peptone Water 006 011 021 044 047 049 055 058 059 062 064 069 075(tmA) 078 086 088 093 094 099 115 122 124 135

Lactose Broth 057

Modified Buffered Peptone Water 021(tmB) 032

Mueller Kauffman 047 055 059 075(tmA) 078 086 121

Rappaport Vassiliadis Broth 021 047 056 057 064 094 121 135

Rappaport Vassiliadis Soya Broth 006 032 044 049 055 058 059 075(tmA) 078 086

Selenite Cysteine Broth 021 094

Tetrathionate Broth 032 057 115

EEM Broth 100

Muller - Kauffman Tetrathionate- Novobiocin 006

S.P.R.I.N.T. Salmonella 007

Salmonella spp. Selective Plating laboratory number

Bismuth Sulfite Agar 057 099

Brilliant Green Agar (BGA) 011 021(tmB) 032 044 047 049 055 056 062 064 086 094

Hektoen Agar 011 057 059 069

Mannitol Lysine Crystal Violet Brilliant Green Agar(MLCB)

044 100 115 124

Rambach Agar 044 078 094

Salmonella/Shigella Agar 011 124 135

Xylose Lysine Desoxycholate (XLD) 006 021 032 047 049 055 057 059 064 069 075(tmA) 078 086 088 093 094 115 122 135

Bactometer 021

Brilliant Green Agar(modified) (PBGA) 006

brilliant green sulfa agar 099

CHROM agar Salmonella 007 058 122

Chromogener-Salmonellen-Agar (Oxoid) 121

Desoxycholate Hydrogen-sulfide Lactose agar 058

DHL, SMID 124

SM ID2 047 075(tmA)

VIDAS-SM ID 062


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Listeria monocytogenes


yes 021 061 063 064 067 068 072 076 078 079 083 089 093 094 099 100 107 109 112 119 121 122 124 125 126 132 138 139

no 009 040 056 062(tmA) 066 082 084 088 090 120 128 133 134

L. monocytogenes Enrichment laboratory number

Buffered Peptone Water 082

Fraser Broth 021 040 056 064 066 072 076 083 088 089 090 094 109 121 125 126 128 130 132 133 138 139

Half Fraser Broth 061 062 064 067 072 078 089 094 107 109 120 121 126 128 132 133 138 139

LEB1/ LEB2 009 079 093 122 124

Listeria PALCAM Selective Enrichment Broth 063 099 112

Listeria Enrichment Broth 100 119

Listeria enrichment broth base 068

TECRA Listeria enrichment broth 134

TSYEB 084

L. monocytogenes Selective Plating laboratory number

Agar Listeria Ottavani + Agosti (ALOA) 061 067 072 078 088(tmB) 089 112 121 130 132 133 138

Oxford Agar 009 021 040 056 062 063 064 072 076 082 083 084 093 094 099 112 119 124 133

PALCAM Agar 021 062 063 064 068 072 079 088(tmA) 089 090 094 099 100 107 109 112 119 120 122 124 132 133 138 139

ELISA perform 134

ALOA + PALCAM Agar 125

CHROM agar Listeria 122 124126

confirmation by vitek system 082

L - rapid mono 112

Listeria Rapid test Oxoid 066

OAA 062

VIDAS LMO2 AFNOR (N0 BIO-12/9-7/02) 128


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E. coli O157


yes 063 078 099 106 114 125 127 138

no 006 009 012 030 036 077 087 094 108 109 136

E. coli O157 Enrichment laboratory number

Brilliant Green Agar (BGA) 109

Buffered Peptone Water 108

EC Broth 030 063 077

Modified Tryptone Soya Broth & Novobiocin 006 009 010 012 036 087 094 099 106 125 127 136 138

mEC-broth with Novobiocin 114

Tryptone Soya Broth 078

Immunomagnetic Separation Used laboratory number

yes 010 012 078 099 106 114 127 138

no 006 009 030 036 063 077 087 094 108 109 125 136

E. coli O157 Selective Plating laboratory number

CR-SMAC 109

CT-SMAC 006 009 010 078 106 108 127 138

Fluorocult 063 087

SMAC 030 063 077 078 094 125 136 138

CHROM agar tm O157 106

Chromagar(CMA) 006

Eosin Methylen Blue agar 114

mHC and TCCSMAC 099

miniVIDAS 012

PCR 036


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APPENDIX VI: Reports and Protocol

FEPAS REPORTS

This FEPAS® report is priced £50 but participants in this assessment receive one copy free of charge.

Participants can download additional copies and check their laboratory number for this and other proficiency tests in which they have participated by logging into the secure area of the FEPAS® website.

Please contact FEPAS® if you require a username and password.

2. FEPAS PROTOCOL

The Protocol [2] sets out how FEPAS® is organised. It gives full details of the statistical procedures used and includes worked examples. Copies can be downloaded from our website.

3. CONTACT DETAILS

FEPAS

CENTRAL SCIENCE LABORATORY Sand Hutton, York

YO41 1LZ UK

Tel: +44 (0)1904 462100 Fax: +44 (0)1904 462111

e-mail: [email protected]

web: www.fepas.com

copy of report109 - gsiクレオス理化学課sid.gsi.co.jp/csl/fapas/report_fepas.pdf ·...

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