Salmonella species occurrence related to coliform counts, and the hygiene and sanitation conditions in ready-to-eat food outlets.

SALMONELLA SPECIES OCCURRENCE RELATED TO COLIFORM

COUNTS AND THE HYGIENE AND SANITATION CONDITIONS IN

READY-TO-EAT FOOD OUTLETS

Marty Linda Hasu

Graduate School

Department of Food Science, Bogor Agricultural University

2009

DECLARATION

I declare that this thesis titled “Salmonella species occurrence related to coliform counts, and the hygiene and sanitation conditions in ready-to-eat food outlets.” was entirely completed entirely by myself with resourceful help from the Department of Food Science, Bogor Agricultural University. Information and quotes which were sourced from journals and books have been acknowledged and mentioned where in the thesis they appear. All complete references are given at the end of the paper.

Bogor, May 2009

Marty Linda Hasu ID: F251078231

ABSTRACT

MARTY LINDA HASU. F2510782341. Salmonella species occurrence related to coliform counts, and the hygiene and sanitation conditions in ready-to-eat food outlets.

Salmonella bacteria species are all pathogenic in nature and are by far one of the groups that cause many food-borne illnesses. The indicator organism, coliforms are mostly used to designate the safety nature of the foods, by indicating the hygiene and sanitation conditions which in turn help to estimate the storability of goods as well. There are many ways to get these microorganisms occur in a food system. One of these is ready-to-eat (RTE) foods (cooked food, fruits and drinks ready made and are set for eating upon customer request). The objectives of this research were to 1) isolate and identifySalmonella species from three ready-to-eat (RTE) food outlets; 2) evaluate the hygiene and sanitation conditions of the ready-to-eat food outlets; 3) establish the coliform counts and relate their occurrence to the hygiene conditions of the three RTE food outlets; 4) determine whether the amounts of coliform counts influence the occurrence ofSalmonella in the samples from RTE food outlets.

The results found occurrence of Salmonella Weltevreden in outlet I from tea towels/dish cloths, food preparation table surfaces, utensils washing water and Salmonella Agona in outlet II from food preparation table surfaces and utensils washing water . The coliform count in outlet I was the highest (total colifoms/all samples 103 MPN/100cm2and fecal coliforms/4.87 x 100 7.01 x 101 MPN/100cm2), followed by outlet II (total coliforms/ranging from 101 - 103 MPN/100cm2and fecal coliforms/3.20 x 100 4.67 x 101 MPN/100cm2), and outlet III (total coliforms/ranging from 101 - 102 MPN/100cm2and fecal coliforms/3.00 x 100 3.00 x 101 MPN/100cm2) having the least number of coliforms. From the hygiene and sanitation inspection it can be said that the cleaner the outlet the lower the coliform count which is the case for outlet III. Outlets with higher coli form counts hadSalmonella isolated.

SUMMARY

MARTY LINDA HASU. F2510782341. Salmonella species occurrence related to coliform counts, and the hygiene and sanitation conditions in ready-to-eat food outlets.

The presence of enteric bacteria like Salmonella and certain coliform groups can be very harmful. Salmonella is responsible for serious and sometimes fatal food poisoning. Coliforms are an indication of whether there has been fecal contamination or the determine the general hygiene and sanitation status of an area; mostly employed to indicate food safety and sanitation conditions of the work areas in general including environment, equipments, utensils, food handlers, and raw materials and the distribution network. Both groups of organisms are of major concern in the food sector. Ready-to-eat (RTE) foods provide a source of possible cause for some of the most dangerous food-borne illnesses. Examples of ready-to-eat foods sold in the RTE food outlets around Bogor Agricultural University area are Fat cakes, doughnuts; corn-on-the-cob, crisp extruded products (Kurupuk), beef, goat meat, fish and chicken, apples, bananas, pears, oranges, grapes and mangoes, soft drinks, juices, and ice pop, Rice is mainly served with the meaty products or vegetarian choices like gado-gado. The number of customers to have one of the outlets serve when filled ranges (outlet I) from 6 8 people; (outlet II) 14 16 people; (outlet III) more than 20 people.

The objectives of this research were to 1) isolate and identifySalmonellaspecies from three ready-to-eat (RTE) food outlets; 2) evaluate the hygiene and sanitation conditions of the ready-to-eat food outlets; 3) establish the coliform counts and relate their occurrence to the hygiene conditions of the three RTE food outlets; 4) determine whether the amounts of coliform counts influence the occurrence of Salmonella in the samples from RTE food outlets.

Detection and isolation of Salmonella sp. were done using the Bacterial Analyisis Methods (BAM - USFDA/CFSAN 2003) whilst determination of which pathogenic Salmonella species were done using serology test. Coliforms were enumerated (quantitative) by the MPN (Most Probable Number) technique. The hygiene and sanitation conditions of ready-to-eat food outlets were evaluated using GMP inspection sheets. All the three activities were inter-related to see if the hygiene and sanitation conditions had an effect on the number of coliforms and the presence of pathogenicSalmonella species. It was found that RTE food outlet I had the highest total coliform count in all four types of samples (total colifoms/all samples 103 MPN/100cm2); it also had the highest fecal coliforms in utensils rinse water (7.01 x 101MPN/100ml), food preparation tables (5.03 x 101MPN/100cm2) and hands of food handlers (4.87 x 100 MPN/100cm2), and outlet II had the highest fecal coliform occurrence in tea towel sample (2.05 x 101MPN/100cm2). Both outlets I and II also were rated low in their GMP inspection scores with higher coliform and fecal contaminations andSalmonella species (S. Weltevreden and S. Agona) were detected.

Recommendation for improving hygiene and sanitation conditions of outlets I and II through training and awareness is vital.

© Copyright of IPB, year 2008 Copyright reserved

1. Forbidden to quote part or all of these writings without including or mentioning the source.

a. Be cited only for educational purposes, research, writing papers, drafting reports, writing criticism or review an issue;

b. Quotation must not harm the affairs of IPB.

2. Prohibit publication and reproduction of part or all of the paper in any form without permission of IPB or the writer.

SALMONELLA SPECIES OCCURRENCE RELATED TO COLIFORM

COUNTS AND THE HYGIENE AND SANITATION CONDITIONS IN

READY-TO-EAT FOOD OUTLETS

MARTY LINDA HASU

THESIS

As Part of the Requirement of Master of Science in Food Sciences

Graduate School

Department of Food Science, Bogor Agricultural University

2009

Title: Salmonella species occurrence related to coliform counts, and the hygiene and sanitation conditions in ready-to-eat food outlets.

Name: Marty Linda Hasu Registration Number: F251078231

Major: Master of Sciences in Food Science

Approved: Advisory Committee

……….. ………. Prof.Dr.Ir.Betty Sri Laksmi Jenie, MSc Dr.Ir.Ratih Dewanti-Hariyadi, MSc

(Chairman) (Member)

Agreed:

Coordinator of Major Dean of Graduate School

………. ………

Dr.Ir.Ratih Dewanti-Hariyadi, MSc Prof. Dr.Ir. Khairil Anwar Notodiputro, MS

ACKNOWLEDGEMENT

Thanks be to God Almighty for guiding, strengthening and for His endless blessings that have seen this research work completed, titled “MARTY LINDA HASU. F2510782341.Salmonella species occurrence related to coliform counts, and the hygiene and sanitation conditions in ready-to-eat food outlets”.

Thank you also to Prof. Dr. Ir. Betty Sri Laksmi Jenie, MSc, and Dr. Ir. Ratih Dewantih-Hariyadi, MSc, supervisory committee for the research work in which invaluable guidance and direction was given for the whole research period.

Special thank you to the Department of Food Science and Srikandi Foundation for the full support given to enable the successful completion of this research. Extended thank you also to all the invaluable lecturing staff members as well as lab and technical staff of Food Science Department who have in one way or another imparted knowledge that I acknowledge with gratitude. All fellow students of Food Science Department and the family of KNB Scholarship group, thank you all for your support and assistance during the course of Masters Degree studies in Bogor Agricultural University. Special mention of close friends Mamihery Ravoniarijaona, Phimmasone Sisouvanh and Amirouche Chikhoune, always providing a helping hand.Further, heartfelt gratitude and thank you to dad, mum, big brother Greg and big sister Julie for all your prayers and endless support, and to Uncle Kelly for all assistance kindly given.

With that, this paper is dedicated to family members and friends, and fellow students; may this study be beneficial for us.

God bless us all.

Bogor Agricultural University. June 2009 MSc. Food Sciences

BIOGRAPHY

The writer, Marty Linda Hasu was born on the 1st of January, 1981 in Port Moresby to Mr Mautaia Hasu and Mrs Mistonka Onne-Hasu from Papua New Guinea. Marty Linda is the second and the last born of two girls.

In 1998, the writer finished Completed Senior High School from Aiyura National High School and continued with Bachelors Degree studies in Food Sciences and graduated in March 2003, from the Papua New Guinea University of Technology. After Bachelors level, the writer won Rotary Youth Leadership award and took up leadership program with Rotary youth tour, Brisbane, Australia. In 2006, Marty Linda Hasu was accepted under the Developing countries partnership program (KNB – Kemitraan Negara Berkembang) awarded by the Indonesian Government to do Masters Degree in Bogor Agricultural University.

LIST OF TABLES ... ii

LIST OF FIGURES ... iii

LIST OF APPENDICES ... iv

1. INTRODUCTION... 1

1.1 Background ... 1

1.2 Objectives and benefits... 2

1.3 Hypothesis... 3

2. LITERATURE REVIEW ... 4

2.1Salmonella... 4

2.1.1 Taxonomy and characteristics ... 4

2.1.2 Gram negative bacteria cell and pathogenic components... 5

2.1.3 Salmonellosis... 7

2.1.4 Occurrences associated with foods... 8

2.2 Coliforms ... 13

2.2.1 Taxonomy and characteristics ... 14

2.2.2 Sources of coliforms ... 14

2.2.3 Coliforms as indicator organisms ... 15

2.2.4 Occurrences: prevalence and epidemiology... 15

2.3 Sources of contamination forSalmonella and coliforms... 16

2.4 Ready-to-eat (RTE) foods and outlets ... 20

3. METHODOLOGY... 21

3.1 Time and place of study... 21

3.2 Types of samples... 21

3.3 Sampling plan... 21

3.4 Criteria for selecting ready-to-eat foods ... 21

3.5 Sampling method... 22

3.5.1 Collecting samples ... 22

3.5.1.1 Utensils washing water ... 22

3.5.1.2 Tea towels ... 23

3.5.1.3 Food preparation tables ... 23

3.5.1.4 Hands of food handlers... 24

3.6 Isolating and identifyingSalmonella species... 24

3.6.1 Non-selective enrichment (resuscitation)... 25

3.6.2 Selective enrichment ... 26

3.6.3 Plating on selective solid media ... 26

3.6.4 Sub-culturing of presumptiveSalmonella ... 26

3.6.5 Biochemical reaction confirmation... 27

3.6.6 Serological somatic (O) tests forSalmonellaserovars ... 28

3.7 Evaluation of hygiene and sanitation conditions... 29

3.7.1 Descriptive inspection... 29

3.7.2 Scoring system... 29

3.8 Method for counting coliforms ... 30

3.8.1 MPN (Most Probable Number) test for coliforms ... 30

3.8.1.1 Preparation of decimal dilutions ... 31

SALMONELLA SPECIES OCCURRENCE RELATED TO COLIFORM

COUNTS AND THE HYGIENE AND SANITATION CONDITIONS IN

READY-TO-EAT FOOD OUTLETS

Marty Linda Hasu

Graduate School

Department of Food Science, Bogor Agricultural University

2009

DECLARATION

I declare that this thesis titled “Salmonella species occurrence related to coliform counts, and the hygiene and sanitation conditions in ready-to-eat food outlets.” was entirely completed entirely by myself with resourceful help from the Department of Food Science, Bogor Agricultural University. Information and quotes which were sourced from journals and books have been acknowledged and mentioned where in the thesis they appear. All complete references are given at the end of the paper.

Bogor, May 2009

Marty Linda Hasu ID: F251078231

ABSTRACT

MARTY LINDA HASU. F2510782341. Salmonella species occurrence related to coliform counts, and the hygiene and sanitation conditions in ready-to-eat food outlets.

Salmonella bacteria species are all pathogenic in nature and are by far one of the groups that cause many food-borne illnesses. The indicator organism, coliforms are mostly used to designate the safety nature of the foods, by indicating the hygiene and sanitation conditions which in turn help to estimate the storability of goods as well. There are many ways to get these microorganisms occur in a food system. One of these is ready-to-eat (RTE) foods (cooked food, fruits and drinks ready made and are set for eating upon customer request). The objectives of this research were to 1) isolate and identifySalmonella species from three ready-to-eat (RTE) food outlets; 2) evaluate the hygiene and sanitation conditions of the ready-to-eat food outlets; 3) establish the coliform counts and relate their occurrence to the hygiene conditions of the three RTE food outlets; 4) determine whether the amounts of coliform counts influence the occurrence ofSalmonella in the samples from RTE food outlets.

The results found occurrence of Salmonella Weltevreden in outlet I from tea towels/dish cloths, food preparation table surfaces, utensils washing water and Salmonella Agona in outlet II from food preparation table surfaces and utensils washing water . The coliform count in outlet I was the highest (total colifoms/all samples 103 MPN/100cm2and fecal coliforms/4.87 x 100 7.01 x 101 MPN/100cm2), followed by outlet II (total coliforms/ranging from 101 - 103 MPN/100cm2and fecal coliforms/3.20 x 100 4.67 x 101 MPN/100cm2), and outlet III (total coliforms/ranging from 101 - 102 MPN/100cm2and fecal coliforms/3.00 x 100 3.00 x 101 MPN/100cm2) having the least number of coliforms. From the hygiene and sanitation inspection it can be said that the cleaner the outlet the lower the coliform count which is the case for outlet III. Outlets with higher coli form counts hadSalmonella isolated.

SUMMARY

MARTY LINDA HASU. F2510782341. Salmonella species occurrence related to coliform counts, and the hygiene and sanitation conditions in ready-to-eat food outlets.

The presence of enteric bacteria like Salmonella and certain coliform groups can be very harmful. Salmonella is responsible for serious and sometimes fatal food poisoning. Coliforms are an indication of whether there has been fecal contamination or the determine the general hygiene and sanitation status of an area; mostly employed to indicate food safety and sanitation conditions of the work areas in general including environment, equipments, utensils, food handlers, and raw materials and the distribution network. Both groups of organisms are of major concern in the food sector. Ready-to-eat (RTE) foods provide a source of possible cause for some of the most dangerous food-borne illnesses. Examples of ready-to-eat foods sold in the RTE food outlets around Bogor Agricultural University area are Fat cakes, doughnuts; corn-on-the-cob, crisp extruded products (Kurupuk), beef, goat meat, fish and chicken, apples, bananas, pears, oranges, grapes and mangoes, soft drinks, juices, and ice pop, Rice is mainly served with the meaty products or vegetarian choices like gado-gado. The number of customers to have one of the outlets serve when filled ranges (outlet I) from 6 8 people; (outlet II) 14 16 people; (outlet III) more than 20 people.

The objectives of this research were to 1) isolate and identifySalmonellaspecies from three ready-to-eat (RTE) food outlets; 2) evaluate the hygiene and sanitation conditions of the ready-to-eat food outlets; 3) establish the coliform counts and relate their occurrence to the hygiene conditions of the three RTE food outlets; 4) determine whether the amounts of coliform counts influence the occurrence of Salmonella in the samples from RTE food outlets.

Detection and isolation of Salmonella sp. were done using the Bacterial Analyisis Methods (BAM - USFDA/CFSAN 2003) whilst determination of which pathogenic Salmonella species were done using serology test. Coliforms were enumerated (quantitative) by the MPN (Most Probable Number) technique. The hygiene and sanitation conditions of ready-to-eat food outlets were evaluated using GMP inspection sheets. All the three activities were inter-related to see if the hygiene and sanitation conditions had an effect on the number of coliforms and the presence of pathogenicSalmonella species. It was found that RTE food outlet I had the highest total coliform count in all four types of samples (total colifoms/all samples 103 MPN/100cm2); it also had the highest fecal coliforms in utensils rinse water (7.01 x 101MPN/100ml), food preparation tables (5.03 x 101MPN/100cm2) and hands of food handlers (4.87 x 100 MPN/100cm2), and outlet II had the highest fecal coliform occurrence in tea towel sample (2.05 x 101MPN/100cm2). Both outlets I and II also were rated low in their GMP inspection scores with higher coliform and fecal contaminations andSalmonella species (S. Weltevreden and S. Agona) were detected.

Recommendation for improving hygiene and sanitation conditions of outlets I and II through training and awareness is vital.

© Copyright of IPB, year 2008 Copyright reserved

1. Forbidden to quote part or all of these writings without including or mentioning the source.

a. Be cited only for educational purposes, research, writing papers, drafting reports, writing criticism or review an issue;

b. Quotation must not harm the affairs of IPB.

2. Prohibit publication and reproduction of part or all of the paper in any form without permission of IPB or the writer.

SALMONELLA SPECIES OCCURRENCE RELATED TO COLIFORM

COUNTS AND THE HYGIENE AND SANITATION CONDITIONS IN

READY-TO-EAT FOOD OUTLETS

MARTY LINDA HASU

THESIS

As Part of the Requirement of Master of Science in Food Sciences

Graduate School

Department of Food Science, Bogor Agricultural University

2009

Title: Salmonella species occurrence related to coliform counts, and the hygiene and sanitation conditions in ready-to-eat food outlets.

Name: Marty Linda Hasu Registration Number: F251078231

Major: Master of Sciences in Food Science

Approved: Advisory Committee

……….. ………. Prof.Dr.Ir.Betty Sri Laksmi Jenie, MSc Dr.Ir.Ratih Dewanti-Hariyadi, MSc

(Chairman) (Member)

Agreed:

Coordinator of Major Dean of Graduate School

………. ………

Dr.Ir.Ratih Dewanti-Hariyadi, MSc Prof. Dr.Ir. Khairil Anwar Notodiputro, MS

ACKNOWLEDGEMENT

Thanks be to God Almighty for guiding, strengthening and for His endless blessings that have seen this research work completed, titled “MARTY LINDA HASU. F2510782341.Salmonella species occurrence related to coliform counts, and the hygiene and sanitation conditions in ready-to-eat food outlets”.

Thank you also to Prof. Dr. Ir. Betty Sri Laksmi Jenie, MSc, and Dr. Ir. Ratih Dewantih-Hariyadi, MSc, supervisory committee for the research work in which invaluable guidance and direction was given for the whole research period.

Special thank you to the Department of Food Science and Srikandi Foundation for the full support given to enable the successful completion of this research. Extended thank you also to all the invaluable lecturing staff members as well as lab and technical staff of Food Science Department who have in one way or another imparted knowledge that I acknowledge with gratitude. All fellow students of Food Science Department and the family of KNB Scholarship group, thank you all for your support and assistance during the course of Masters Degree studies in Bogor Agricultural University. Special mention of close friends Mamihery Ravoniarijaona, Phimmasone Sisouvanh and Amirouche Chikhoune, always providing a helping hand.Further, heartfelt gratitude and thank you to dad, mum, big brother Greg and big sister Julie for all your prayers and endless support, and to Uncle Kelly for all assistance kindly given.

With that, this paper is dedicated to family members and friends, and fellow students; may this study be beneficial for us.

God bless us all.

Bogor Agricultural University. June 2009 MSc. Food Sciences

BIOGRAPHY

The writer, Marty Linda Hasu was born on the 1st of January, 1981 in Port Moresby to Mr Mautaia Hasu and Mrs Mistonka Onne-Hasu from Papua New Guinea. Marty Linda is the second and the last born of two girls.

In 1998, the writer finished Completed Senior High School from Aiyura National High School and continued with Bachelors Degree studies in Food Sciences and graduated in March 2003, from the Papua New Guinea University of Technology. After Bachelors level, the writer won Rotary Youth Leadership award and took up leadership program with Rotary youth tour, Brisbane, Australia. In 2006, Marty Linda Hasu was accepted under the Developing countries partnership program (KNB – Kemitraan Negara Berkembang) awarded by the Indonesian Government to do Masters Degree in Bogor Agricultural University.

LIST OF TABLES ... ii

LIST OF FIGURES ... iii

LIST OF APPENDICES ... iv

1. INTRODUCTION... 1

1.1 Background ... 1

1.2 Objectives and benefits... 2

1.3 Hypothesis... 3

2. LITERATURE REVIEW ... 4

2.1Salmonella... 4

2.1.1 Taxonomy and characteristics ... 4

2.1.2 Gram negative bacteria cell and pathogenic components... 5

2.1.3 Salmonellosis... 7

2.1.4 Occurrences associated with foods... 8

2.2 Coliforms ... 13

2.2.1 Taxonomy and characteristics ... 14

2.2.2 Sources of coliforms ... 14

2.2.3 Coliforms as indicator organisms ... 15

2.2.4 Occurrences: prevalence and epidemiology... 15

2.3 Sources of contamination forSalmonella and coliforms... 16

2.4 Ready-to-eat (RTE) foods and outlets ... 20

3. METHODOLOGY... 21

3.1 Time and place of study... 21

3.2 Types of samples... 21

3.3 Sampling plan... 21

3.4 Criteria for selecting ready-to-eat foods ... 21

3.5 Sampling method... 22

3.5.1 Collecting samples ... 22

3.5.1.1 Utensils washing water ... 22

3.5.1.2 Tea towels ... 23

3.5.1.3 Food preparation tables ... 23

3.5.1.4 Hands of food handlers... 24

3.6 Isolating and identifyingSalmonella species... 24

3.6.1 Non-selective enrichment (resuscitation)... 25

3.6.2 Selective enrichment ... 26

3.6.3 Plating on selective solid media ... 26

3.6.4 Sub-culturing of presumptiveSalmonella ... 26

3.6.5 Biochemical reaction confirmation... 27

3.6.6 Serological somatic (O) tests forSalmonellaserovars ... 28

3.7 Evaluation of hygiene and sanitation conditions... 29

3.7.1 Descriptive inspection... 29

3.7.2 Scoring system... 29

3.8 Method for counting coliforms ... 30

3.8.1 MPN (Most Probable Number) test for coliforms ... 30

3.8.1.1 Preparation of decimal dilutions ... 31

3.8.1.3 Recording results... 32

4. RESULTS AND DISCUSSION ... 33

4.1Salmonella... 33

4.2 Hygiene and sanitation conditions ... 38

4.2.1 Employee hygiene and behavior... 38

4.2.2 Building and facility design and plan ... 39

4.2.3 Environmental sanitation ... 41

4.2.4 Processing sanitation ... 42

4.2.5 Water source and usage... 44

4.2.6 Overall hygiene... 46

4.3 Coliforms ... 48

4.3.1 Total coliform count ... 48

4.3.1.1 Utensils washing water... 48

4.3.1.2 Tea towels ... 49

4.3.1.3 Food preparation tables ... 50

4.3.1.4 Hands of food handlers... 51

4.3.2 Fecal Coliforms ... 52

4.3.2.1 Utensils washing water ... 53

4.3.2.2 Tea towels ... 54

4.3.2.3 Food preparation tables ... 55

4.3.2.4 Hands of food handlers... 56

4.4 Comparison of coliforms and hygiene and sanitation conditions ... 56

4.5 Comparison coliform count andSalmonella occurrence... 57

5. CONCLUSION... 58

6. RECOMMENDATION... 58

7. REFERENCES ... 59

8. APPENDICES ... 69

Limits for the growth ofSalmonella under otherwise optimal conditions, adapted from International Commission

on Microbiological Specifications for Foods (1996) ... 4 Distribution of the 10 most commonSalmonella

serovars from the different reservoirs, Thailand... 11 Grading criteria using the modified Badan POM criteria ... 30 Number of positiveSalmonellaisolated ... 34 Serology test results forSalmonella serovars... 34 MPN counts for total coliforms ... 48 MPN counts for fecal coliforms... 53

LIST OF FIGURES

Pathogenesis of salmonellosis ... 8 Fecal oral routes of transmission of food-borne intestinal pathogens... 16 Iceboxes used during sampling... 22 Diagram of swabbing action... 23 Diagram of the procedure for detection ofSalmonella... 25 Summary of MPN (Most Probable Number) methods

for total coliforms, fecal coliforms andE.coli... 31 Employee hygiene and sanitation ... 39 Building and facilty design and plan... 41 Environmental sanitation... 42 Processing sanitation... 44 Water source and usage... 46

LIST OF APPENDICES

Biochemical and serological reactions ofSalmonella... 69 Criteria for discarding non-Salmonellacultures... 70 Inspection check sheets for RTE food outlets I, II, and III ... 71 Employee hygiene and behavior... 85 Building and facilities' design and plan ... 85 Environmental Sanitation ... 86 Processing sanitation... 86 Water source and usage... 86 Grading criteria using the modified Badan POM criteria ... 87 Photos of the ready-to-eat food outlets ... 87 MPN index and 95% confidence limits for various combinations of

positive tubes in a 3 tube dilution series using inoculums quantities

of 1, 0.1, and 0.01 g (ml) ... 89 For 3 tubes each at 0.1, 0.01, and 0.001 g inocula, the MPNs per gram

and 95 percent confidence intervals... 90 Least Significant difference between RTE food outlets for total coliforms... 91 Least Significant difference between RTE food outlets for fecal coliforms ... 91 List of equipment and materials... 92

1. INTRODUCTION

1.1 Background

In developing countries, drinks, meals (ready-to-eat) and snacks sold by food vendors are widely consumed by millions of people (FAO, 1988). These foods provide an affordable source of nutrients to many sectors of population (Ohiokpehai, 2003). These ready-to-eat (RTE) foods are well appreciated by consumers, because of their taste, low price and availability at the right time (Barro et al., 2002b; Canet and Diaye, 1996). However, the conditions at ready-to-eat foods and vending raise many concerns for consumers health (Bryanet al., 1988; Mosupye and van Holy, 1999).

In most cases, running water is not available at vending sites, hands and dish washing are usually done in one or more buckets, and sometimes without soap. Waste waters and garbages are discarded nearby, providing nutrients for insects and rodents. Some of the foods are not efficiently protected against flies which may carry foodborne pathogens. Safe food storage temperatures are rarely applied to most of the small ready-to-eat (RTE) food outlets (Bryanet al., 1988). In addition, there are potential health risk associated with initial contamination of foods by pathogenic bacteria as well as subsequent contamination by vendors during preparation and through post-cooking handling and cross-contamination (Bryanet al., 1988).

Contamination is a very important aspect as this is the mode that most unwanted microorganisms may be transmitted onto food products. Unwanted microorganisms may access food processing environments through raw material, personnel or equipment and utensils, through leakage and openings in buildings or through pests, and some pathogens may even become established in the food processing area and form niches where they can survive for long periods of time (Reij et al. 2003). Many of these microorganisms occur naturally in general environments, and may be transmitted onto foods. Furthermore, contamination via air can occur through dust particles or via aerosols which are formed especially when contaminated surfaces, floors or drains are sprayed with high pressure-jets, resulting in formation of droplets that can be suspended in the air (Aantrekker et al. 2003). Water is also a vehicle for transmission of many agents of diseases (Kirbyet al. 2003).

Food service employees and others need to be educated about effective food safety, including purchasing of raw materials, storing, handling of foods properly, cooking foods to proper temperatures, using effective hand-washing practices, keeping food contact surfaces clean and sanitized preventing cross-contamination (Fryback 1998).

The organisms of interest in this research are Salmonella and coliforms. With respect to Salmonella, it is a major foodborne pathogen and causes a disease known as salmonellosis. Coliforms are indicator organisms; they normally enlighten to the inspectors what state of hygiene, or sanitation condition is shown for the work area. Occurrence of coliforms could be an indication that disease-causing microorganisms like Salmonella may likely be present as well. Hygiene and sanitation checks are carried out to compare with microbial load especially of the indicator groups.

1.2. Objectives and benefit of this research

The objectives of this research were to 1) isolate and identifySalmonellaspecies from three ready-to-eat (RTE) food outlets; 2) evaluate the hygiene and sanitation conditions of ready-to-eat food outlets; 3) establish the coliform counts and connect their occurrence to the hygiene conditions of the three RTE food outlets; 4) find if the amounts of coliform counts influence the occurrence of Salmonella species in the samples from RTE food outlets.

Salmonella being a common foodborne pathogen if present in the samples from the three RTE food outlets would be an indication of possible contamination to the foods sold in those outlets. It would be beneficial to establish the occurrence of this pathogen so as to enlighten to people that there are risks to consuming RTE foods however with proper work practices like cleanliness and neatness, treatment of infected workers, and good employee hygiene would help to reduce the possibility of its presence in RTE foods. Many a case ofSalmonella related food poisoning has been reported in the world, and developing countries have a much higher risk. This may be due to much of the systems enabling safety for populations being not well-established due to financial constraints or lack of information and educating people. Issues of improper sanitation increase the risk and so indicator organisms such as coliforms are a successful find from scientists who developed this useful system for quick assessment of how safe a product may be due to the presence of these indicator organisms.

This research hence was carried out to determine if Salmonella species were present in the ready-to-eat food outlets. Also, presence and count of coliforms using MPN technique in these outlets would draw attention to all the sanitation conditions of the outlets. With the data obtained awareness and improvements of ready-to-eat (RTE) products can be carried out with the advantageous effects on producing safer RTE food outlets by keeping the work areas clean, neat and attractive for the customers; more people would believe in safe RTE food products.

1.3. Hypothesis

1. There areSalmonellaspecies present in the three ready-to-eat food outlets from the four possible contaminated sources (utensils washing water, tea towels, food preparation tables and hands of food handlers.)

2. High coliform counts where hygiene and sanitation conditions are low/poor. 3. Salmonella species are present in outlets where coliform counts are high.

2. LITERATURE REVIEW

2.1Salmonella

2.1.1 Taxonomy and Characteristics

Salmonellae are motile, non-spore forming, Gram- negative, rod-shaped bacteria that belong to the family Enterobacteriaceae. The molecular % of the G + C (Guanine and Cytosine) content in their DNA is 50 53 (Jay, 2000). There are approximately 2,400 Salmonella serovars, which are classified under two species, S. enterica andS. bongori (Jayet al., 2005).

Salmonella grows on a large number of culture media, and produce visible colonies within 24 hours at about 37 °C. The optimum pH growth for this organism is around neutrality (6.6 8.2) with values above 9 and below 4 are bacteriocidal. It grows at high water activity, below 0.94 growth is slowed and even inhibited (Prost and Riemann 1967). The organism cannot tolerate high salt concentrations; above 9% of a product composition salt concentration is considered bacteriocidal. Its temperature range for growth is 4 to 45°C (MacDonald, and Matney. 1963). With respect to destruction all Salmonella are readily destroyed at milk pasteurization temperatures.

Table 1.0 Limits for the growth ofSalmonella under otherwise optimal conditions, adapted from International Commission on Microbiological Specifications for

Foods (1996). Parameter (other

conditions being optimal)

Minimum Maximum

Temperature ( C) 5.2 (most serotypes will not grow at <7.0) 46.2 pH 3.8 (most serotypes will not grow below 4.5) 9.5

Water activity 0.94 >0.99

Source: Chris and Kyriades 2002.

The bacteria can catabolize D-glucose and other carbohydrates with the production of acid and gas. The organism species are oxidase negative and catalase negative, grow on citrate as a sole carbon source, generally produce carbon sulfide, decarboxylase lysine and ornithine and do not hydrolyse urea. Many of these traits form the presumptive basis for the biochemical identification ofSalmonella isolated (Doyle, Beuchat and Montville 2001).

2.1.2 Gram Negative bacteria cell and pathogenic components

Gram negative bacteria possess several distinct surface layers that can enhance their pathogenicity. These layers are flagella, capsules and cell wall components. In figure 1, general bacterial that is of pathogenic nature is portrayed.

Figure 1. General cell structure of bacteria. Source: Fix 1997 2009.

Flagella are the organs of motility. Flagella are composed of flagellins (proteins) that make up the long filament. This filament is connected to a hook and rings that anchor the flagella in the cell wall. In Gram-positive bacteria, there are two rings attached to the cytoplasmic membrane; in Gram-negative cells, an additional two rings are found in the outer membrane. Flagella may be up to 20 µm in length. Some bacteria possess a single polar flagellum (monotrichous), others have several polar flagella (lophotrichous), others have several flagella at each end of the cell (amphitrichous), and still others have many flagella covering the entire cell surface (peritrichious). Flagella may serve as antigenic determinants (e.g. the H antigens of Gram-negative enteric bacteria) (Fix, 1997 2009).

Capsules are types of surface layer composed primarily of high molecular weight polysaccharides. If the layer is strongly adhered to the cell wall, it is called a capsule; if not, it is called a slime layer. These layers provide resistance to phagocytosis and serve as antigenic determinants, the Vi or K antigen. The production of capsules is genetically and phenotypically controlled.

The cell wall is the basis for classification of bacteria according to the Gram stain. Gram-positive bacteria have a thick layer of peptidoglycan external to the cytoplasmic membrane. In contrast, Gram-negative bacteria have a thin layer of peptidoglycan located between the cytoplasmic membrane and a second membrane called the outer membrane. This region is known as the periplasmic space. Peptidoglycan is a polymer of alternating N-acetylmuramic acid (NAM) and N -acetylglucosamine (NAG). Long strands of this alternating polymer may be linked by L-alanine, D-glutamic acid, L-lysine, D-alanine tetrapeptides to NAM. Gram-positive cells have a much more highly cross-linked peptidoglycan structure than Gram-negative cells. Peptidoglycan is also the "target" of antimicrobial activity. For example, penicillins interfere with the enzymes involved in biosynthesis of peptidoglycan while lysozyme physically cleaves the NAM-NAG bond. Lipopolysaccharides (LPS) are found only in Gram-negative bacteria. These structures are composed of lipid A, which binds the LPS in the outer membrane and is itself the endotoxic portion of the molecule. The polysaccharide moiety appears on the cell surface, serving as an antigenic determinant ("O antigen") (Wheelis 2007).

Somatic O antigens in Gram negative bacteria are situated in the outer membrane of the cell and are attached to lipids, commonly known as lipid A. Serological tests use somatic O to determine the bacterial serovars specifically being tested using antibodies of presumed organisms suspected to be present to specifically get identified.

The presence of an outer membrane and the possession of only few peptidoglycan layers in the cell wall distinguish negative bacteria from Gram-positive ones (figure 2). Lipids covalently linked to proteins called lipoproteins are the molecules that bind the peptidoglycan to the outer membrane. The peptidoglycan is located in the periplasm, a space filled with fluid located between the plasma membrane and the outer membrane. A high amount of degradative enzymes and transport proteins are found in the periplasm. Unlike Gram-positive cell walls, we cannot find teichoic acids in the Gram-negative cell walls. In addition, the cell walls of Gram-negative bacteria are more prone to mechanical breakage because of the low amount of peptidoglycan. (Shagam 2006)

2.1.3 Salmonellosis

In humans, salmonellosis usually takes the form of a self-limiting food poisoning (gastroenteritis), but occasionally manifests as a serious systemic infection (enteric fever) which requires prompt antibiotic treatment. In addition, salmonellosis causes substantial losses of livestock (WHO Fact sheet, 2005). The disease occurs as a result a large number of the Salmonella organism (infectious dose) entering the host through contaminated products at farm levels and/or ranch levels, industry levels or household levels but the infectious dose may be lower for certain susceptible groups such as children, the elderly and the immuno-compromised. The number of cells required for the onset of salmonellosis is said to be in the order of 107-109/g (Jayet al., 2005). Figure 1 shows the pathway of spreading, and where the gastroenteritis and diarrhea occur.

The most common symptoms of salmonellosis include sudden onset of abdominal cramps and nausea followed by diarrhea (sometimes bloody), fever, and sometimes vomiting (Jay et al., 2005). Symptoms normally last for 2 to 3 days in healthy individuals, however, children younger than 1 year of age, persons over 50 years of age, and those who are immune compromised, are most susceptible to salmonellosis, and experience the more severe symptoms (Jayet al., 2005, Darwin and Miller, 1999). Attack rates for Salmonella infection are highest in those younger than 5 years of age and older than 70 years of age and those with underlying immunosuppressive conditions. Dehydration may be severe, especially among infants and the elderly, and invasive disease may occur. The infection may also present as septicemia causing suppurative lesions throughout the body, pneumonia, osteomyelitis, or meningitis, or it can be present as an abscess, arthritis or cholecystitis.

Figure 2. Pathogenesis of salmonellosis. Source: (Gianella 1979)

Two types of salmonellosis result because of consuming foods contaminated with Salmonella bacteria: non-typhoidal and typhoidal salmonellosis. Almost all serovars are pathogenic and belong to the non-typhoidal causing salmonellosis, in the form of food poisoning with no enteric fever, except the serovarsS.Typhi,S.Paratyphi A,S. Paratyphi B, and S.Paratyphi C. These three serovars bring about the commonly known typhoid and paratyphoid fevers in humans. S.Typhi initiates typhoid, whilst S.Paratyphi A, B, and C initiate paratyphoid fever; Paratyphoid fever is a systemic disease, caused by having symptoms similar to those of typhoid fever, but they are milder and the case fatality rate is much lower (Maskalyk 2003).

2.1.4 Occurrences associated with foods

Salmonellosis outbreaks have occurred from a variety of foods including poultry, meats, eggs, milk products, fruit juice, fish, shrimp, frog legs, yeast, coconut, sauces and salad dressing, cake mixes, breakfast cereal, cream-filled desserts and toppings, dried gelatin, peanut butter, cocoa, chocolate, and dried spices. The incidence ofSalmonella is much higher in raw agricultural products (e.g. raw eggs, or uncooked poultry or meat) than in cooked or processed food products. However, Salmonella can occur in other foods as a result of cross-contamination with raw foods, or from contamination from humans, animals, birds, or reptiles. Furthermore, due to the microorganisms ability to survive in wide range of environments, Salmonella has been found in dry and dehydrated foods (e.g. cocoa, chocolate, dry milk, spices, and cereal products) and in more acid food products (e.g. non-pasteurized orange juice). Thus, preventative

measures are extremely important at all food handling and processing steps (Schneider et al2003).

Several research studies carried out in Indonesia have foundSalmonella in foods. Rusyanto (2005) isolatedSalmonella from shrimp samples as well as from the sediments and water in which the shrimps were obtained from. Serological tests confirmed several serovars including S. Lexintgon, S. Kirkee, S. Hadar, S. Infantis, and S. Paratyhi B; amongst them S. Paratyphi B causes typhoidal salmonellosis whilst the others cause non-typhoidal salmonellosis. Agustin (2004) isolatedS. Weltevreden from fresh lettuce sold in traditional markets around Bogor area. Out of 50 samples analyzed, 2 samples were serologically confirmed to containSalmonella Weltevreden.

Other studies by Ruslan (2003) and Susilawati (2002) also detected Salmonella in vegetables, however no serological tests was done to identify the serovars present. Susilawati (2002) studied bean sprouts, cabbages, carrots and long green beans; these fresh vegetables were from the farms and traditional markets. The study found that almost 80% of the raw and fresh vegetables were Salmonella positive. The contamination of these vegetables were thought to have been from contaminated irrigation water, washing water and other fecal contamination most probably from fertilizers.

There have been and still will be cases, and outbreaks as a result of Salmonella species inflictions due to consuming contaminated foods, or being passed on from infected persons or animals; a few outbreaks and cases are reported in this paper. The latest of outbreaks due to one of the strains of Salmonella bacteria (S. Typhimurium) was linked to peanut butter in the United States (CDC 2009).

Another outbreak reported in 2008 by Food and Drug Administration (USA) that 145 to 150 people in 16 states were sickened by salmonellosis which was linked to tomatoes being the vegetable containing the pathogen. It was caused by a rare strain of Salmonella Saintpaul; 25 people were hospitalized but no deaths reported (Weise 2008). Also in USA the same year (2008) an outbreak reported to have been caused by S. Agona was linked to cereal from Malt-O-Meal unsweetened Puffed Rice Cereals and unsweetened Puffed Wheat Cereals. Fifteen states identified 28 ill persons infected with same genetic fingerprint ofSalmonella Agona. Inflicted persons with the outbreak strain had been identified from Colorado (1), Delaware (2), Illinois (1), Maine (4),

Massachusetts (2), Michigan (1), Minnesota (1), North Dakota (1), New Hampshire (2), New Jersey (5), New York (3), Ohio (1), Pennsylvania (2), Rhode Island (1), and Vermont (1) (FDA 2008).

Outbreaks were also associated with alfalfa sprouts in Norway, Denmark and Finland from July to October 2007. Nineteen cases had been reported in Norway, nineteen in Denmark, and seven in Finland. The patient s ages ranged from 18 to 83 years (median age 34 years). Thirty-five cases were female and 10 male. The serovar isolated wasS.Weltevreden (Emberlandet al2007).

In August 1999 in Okinawa, 30 persons suspected of food poisoning visited the hospital having major symptoms included diarrhea (96%, average 21 times), fever (85%, average 39 °C), abdominal pain (86%), headache (58%), nausea (28%) and vomiting (25%). Two days earlier on they had participated in a party and consumed goat meat and soup. Microbial tests conducted found that Salmonella Weltevreden was present in stools of 4 of the patients, raw goat meat, and goat meat soup. Meat and environmental samples at the slaughter house also yielded where the meat was from also yielded S. Weltevreden (Kudaka 2000).

In Bangkok, Thailand, from 1993 to 2002, a total of 70, 235 isolates received were confirmed asS. enterica and serotyped by theSalmonella and Shigella diagnostic laboratories. Serotyping was done for 44,087 Salmonella isolates from humans and 26,148 from other sources from 1993 through 2002. Samples were obtained from humans, ducks, chicken, seafood, other food products, and water for all 10 years. Table 2.0 shows the species that were isolated. It can be noted thatS. Weltevreden was present in all the samples except in frozen chicken; S. Agona was present in all the samples except in frozen seafoods (Bangtrakulnonthet al2003).

Table 2.0 Distribution of the 10 most commonSalmonella serovars from the different reservoirs, Thailanda

Reservoir and no. of isolates (% in brackets)

Salmonella serovar Humans _chickenFrozen _seafoodFrozen Frozen_duck Other food_products Water

Weltevreden 5,491 (12.5) — 265 (26.3) 320 (12.0) 457 (6.6) 143 (14.5) Enteritidis 5,010 (11.4) 2,901 (19.9) 14 (1.4) 309 (4.5) 22 (2.2)

Anatum 3,263 (7.4) 423 (2.9) 20 (2.0) 1,177 (17.0) 113

(11.5)

Derby 2,889 (6.6) 20 (2.0) 370 (5.3) 71 (7.2)

1, 4, 5, 12:i:-ssp.I 2,804 (6.4)

Typhimurium 2,322 (5.3) 12 (1.2) 198 (2.9)

Rissen 2,319 (5.3) 21 (2.1) 712 (10.3) 93 (9.5)

Stanley 1,688 (3.8) 20 (2.0) 279 (10.4)

Panama 1,474 (3.3) 41 (1.5) 254 (3.7) 47 (4.8)

Agona 1,096 (2.7) 452 (3.1) 80 (3.0) 273 (3.9) 39 (4.0)

Paratyphi B var Java

1037 (7.1)

Hadar 1,357 (9.3) 21 (2.1) 263 (9.9) 439 (6.3)

Virchow 863 (5.9) 249 (3.6) 27 (2.7)

Schwarzengrund 565 (3.9)

Emek 359 (2.5)

Blockley 676 (4.6)

Amsterdam 368 (2.5) 103 (3.9)

Seftenberg 49 (4.9) 86 (3.2)

Lexington 47 (4.7) 35 (3.6)

Newport 100 (3.7)

Tennessee 77 (2.9)

Chester 171 (6.4)

London 22 (2.2)

Other 15,824 (35.9) 5,558 (38.2) 518 (51.4) 1,150 (43.1)

2,490 (35.9) 372 (37.8)

Total 44,087 14,559 1,007 2,670 6,928 984

a _{, not among the top 10 serovars.}

A few years back, a Salmonella outbreak in England and Wales, which affected 37 people, many of which were children, was said to be caused by Cadbury chocolates that were contaminated with Salmonella serotype Montevideo (Times online, 2006). As a consequence, the Food Standards Agency (FSA) ordered Cadbury s to remove 250 tons of chocolates from shop shelves and warehouses (Daily Mail, 2006). It is quite clear that such actions (recall and disposal of chocolates) would have significant economic consequences.

One other outbreak which affected more than 350 people in Britain, occurred in 2004, and was linked to Salmonella serotype Newport contaminated lettuce from fast food and catering establishments (Daily Mail, 2004). These and other cases of salmonellosis have enforced a more stringent attitude towards food products. Regulation tests conducted during March 2005 to July 2006 by the FSA found that one out of every 30 boxes of eggs from those imported into the UK, tested positive for Salmonella contamination (ElAmin, 2006c). The highest prevalence of contaminated eggs was found to originate from Spain, with S. enteritidis being the most commonly isolated strain. The European Commission prompted efforts to minimize contamination by Salmonella and other foodborne pathogens. These included compulsory vaccination of flocks in countries such as Spain and France, which have the highest prevalence of contaminated birds. Secondly trade bans of eggs with high levels of Salmonella were also proposed. Such actions have been effective in reducing the number of cases of salmonellosis in the 25 EU states, which was reported at 192,703 cases during 2004 (ElAmin, 2006c).

A wide variety of foods have been implicated in outbreaks of illness caused by many different serotypes of Salmonella: raw meats, poultry, eggs, milk and dairy products, fish, shrimp, frog legs, yeast, coconut, sauces and salad dressing, cake mixes, cream-filled desserts and toppings, dried gelatine, peanut butter, cocoa, and chocolate. VariousSalmonellaserotypes have long been isolated from the outside of eggshells. The present situation with S. Enteritidis is complicated by the presence of the organism inside the egg, in the yolk. This and other information strongly suggest vertical transmission, i.e., deposition of the organism in the yolk by an infected layer hen prior to shell deposition. Foods other than eggs have also caused outbreaks of S. Enteritidis disease. Salmonella still is the most frequently recorded pathogen in the production

chain of food of animal origin. At present the predominant serotypes are S. Enteritidis andS. Typhimurium. This is true especially considering the most important meats from pig and poultry. In areas such as Scandinavia measures against this pathogen have been traditionally more thoroughly endeavored, finally resulting in a lower prevalence of Salmonella in these 122 countries compared to Continental Europe. Whatever the Salmonella serotype, effective controls for minimizing/eliminating the hazard of Salmonella from foods involve control of the following steps: raw materials, personal and environmental hygiene, process conditions, post-process contamination, retail and catering practices, consumer handling.

Other studies have shown the occurrence of Salmonella species in foods due to improper hygiene and sanitation processes. Salmonella serotype Hartford and Salmonella serotype Gaminara were isolated from unpasteurized orange juice (Cooket al 1998). Regardless of the environmental source and means of contamination, once Salmonella was introduced into the processing plant, inadequate cleaning and sanitization of processing equipment probably contributed to production of contaminated juice. The presence of a specific fecal indicator organism in all samples of orange juice tested indicates improper sanitation in the processing plant. The identification of the same subtype of a rare Salmonella serotype (Gaminara) in juice produced during a 3-month span (May- July) suggests that there was an ongoing source ofSalmonellawithin the plant (Cooket al 1998).

Ditjen POM (Pengawasan Obat dan Makanan), a body that enforces food and medicinal drug safety in Indonesia set the standard ((RI. No. 03726/B/SL/V1) for foods to be free fromSalmonella (zeroSalmonella) in 25g of samples analyzed. This is also the same requirement by USFDA (United States Food and Drug Administration) (USFDA/CFSAN 2007), that all foods analyzed (25g samples) should be free from Salmonella).

2.2 Coliforms

2.2.1 Taxonomy and characteristics

There are 16 species of total coliform found in soils, plants and in animal and human waste. Coliforms are defined as all aerobic and facultatively anaerobic, gram negative, asporogenous rods able to ferment lactose with the production of acid and gas.

With the exception of Proteus, these organisms ferment lactose, which is a useful characteristic for differentiating them from Salmonella and Shigella. These include species whose habitat is intestinal or non-intestinal (soil, water) and may include: Escherichia coli,Aeromonas hydrophila, Enterobacter cloacae, Klebsiella pneumoniae and Citrobacter species (Clark and Pagel 1977).

The total coliform group of bacteria are an aerobic or facultatively anaerobic, gram-negative, non-sporeforming, rod-shaped bacteria that ferment lactose with gas production in 24 to 48 hours. Total coliform bacteria are discharged in high numbers in animal and human feces, but not all total coliform bacteria are necessarily of fecal origin. These indicator bacteria have been found to be useful for determining safe drinking and recreational waters (Entry and Farmer 2001).

A subgroup of total coliform, called fecal coliform bacteria, is different from the total coliform group because they can grow at higher temperatures (normall around 45°C) and are found only in the fecal waste of warm-blooded animals. There are six species of fecal coliform bacteria found in animal and human waste.E. coliis one type of the six species of fecal coliform bacteria. A rare strain ofE. colithat you may have seen in the news can cause potentially dangerous outbreaks and illness. This strain is calledE. coli0157 (Bureau of Environmental Health, 2004). Fecal coliform bacteria are more thermo-tolerant. Fecal coliform bacteria are bacteria that originate from intestinal tracts of homothermic animals and are used to determine fecal contamination of samples especially water. This group of bacteria has been found to have an excellent positive correlation with fecal contamination of water from warm-blooded animals (Greenberget al., 1992).

2.2.2 Sources of coliforms

Animal feces contain coliform bacteria, microorganisms that inhabit the intestines of warm-blooded animals. Many coliform bacteria are also found on plants and in soil and water. Coliform bacteria are not pathogens themselves, but their presence indicates the possibility of finding pathogens. In contrast, fecal coliform bacteria such as Escherichia coli are found in feces, and their presence in drinking water indicates fecal contamination. E. coli can also be a pathogen itself, so if E. coli is found in drinking

water there is a good chance that other pathogens are present, too (State of Winsconsin 199 2009).

2.2.3 Coliforms as indicator organisms

Detection of coliforms is used as a general indicator of sanitary and hygiene conditions in the food-processing environment. Fecal coliforms remain the standard indicator of choice for shellfish and shellfish harvest waters; and E. coli is used to indicate recent fecal contamination or unsanitary processing.

Coliforms may be transferred onto the raw material through various means; however food-handlers could be the most important source for contaminating the raw material and processing equipment. Other means include contaminated raw materials, via air currents, insects and rodents, and water. Since the presence of these microorganisms may give an impression of the hygienic conditions under which the plant is operating, to reduce or eliminate these microorganisms there is a need for proper application of hygiene and sanitation procedures. Cleaning contact surfaces with approved food standard detergents, and disinfectants may greatly reduce the load of coliforms. Food handlers must was hands thoroughly with suitable detergent, and wear clean attire, and be hygienically presentable. Furthermore, good work practices and behaviors are also of great importance.

Fecal coliform (FC) and Escherichia (E. coli) bacteria are found in greater quantities than total coliform in animal fecal matter. If fecal coliforms or E. coli is detected along with total coliforms in drinking water, there is strong evidence that sewage is present; therefore, a greater potential for pathogenic organisms exists (Department of environmental quality, Idaho).

2.2.4 Occurrences: prevalence and epidemiology

Not all coliforms are pathogenic, those outbreaks and cases that occur are mainly from the fecal group of coliforms specifically the E.coli strains; one common deadly strain isE.coli O157:H7. There are many outbreaks and cases due to this deadly E.colistrain, some of these are presented in this work.

A multistate outbreak linked to Topp s brand ground beef patties in October 2006 reported 40 cases but no deaths reported. The ages of patients ranged from 1 to 77

years; 50% were between 15 and 24 years old (only 14% of the US population is in this age group). Another outbreak of E.coli O157:H7 occurred in December 2006 with 71 persons falling ill by consuming contaminated lettuce at taco Bell restaurant in USA. Among the 71 persons with illness, 53 (75 %) were hospitalized and 8 (11%) developed a type of kidney failure called hemolytic-uremic syndrome (HUS). Another recent outbreak in 2007 reported 10 ill persons due to consuming contaminated pizza. The age of ill persons ranged from 1 to 65 years with a median age of 9; 53% of ill persons were female. At least 8 people had been hospitalized, and 4 had developed a type of kidney failure known as hemolytic-uremic syndrome, or HUS. No deaths had been reported.

2.3 Sources ofSalmonella and Coliform bacteria contaminations

The primary source of Salmonella is the intestinal tract of humans and animals such as farm animals, birds, reptiles and sometimes insects (Jay et al., 2005). Transmission of the organism, which is excreted in faeces, may occur via insects that contaminate food or water, or by the unsanitary handling of foods by food handlers (Jay et al., 2005). The ingestion of Salmonella contaminated foods result in salmonellosis or Salmonella poisoning, which consists of a variety of disease syndromes: bacteremia, focal infections, enteric fever and enterocolitis, the most common of all illnesses (Darwin and Miller, 1999).

Figure 3. Fecal – oral routes of transmission of food-borne intestinal pathogens. The direction is from bottom to top.

Source: (Jay, 2000)

MOUTH

Toddler objects Foods & utensils

Edible foods

Fingers _{Animals and insects}

Water

Recontamination of otherwise sound products through contaminated surfaces has been observed in many cases and is a major issue. For example, unclean, insufficiently or inadequately cleaned pieces of equipment have been identified as the sources of the pathogens are numerous. An interesting aspect is the impact of the micro-flora residing on food contact surfaces of processing equipment used in slaughterhouses, for which the transfer of pathogens such as S. aureus, L. monocytogenes or Salmonella during slaughtering has been demonstrated (Adams and Mead, 1983; Rorvik et al., 1995;Giovannacci et al., 2001). An important element in the investigation of recontamination is the identification of transfer mechanisms. The impact of direct airborne recontamination of products is frequently over-emphasized. It is probably limited to few product categories such as beverages, refrigerated dairy and culinary (products where multiplication can occur) or to products with very low viable counts, such as dry infant formulae. Air filtration is usually performed in plants manufacturing products for which a high probability of airborne recontamination has been identified. Airborne microorganisms are usually associated with dust particles or water droplets (aerosols) and transmission is occurring due to airflow. Distribution of pathogens through aerosols is probably more important than through dust. A more systematic approach to investigating transmission via air that is suitable for modeling has been started (Aantrekker, 2002; Aantrekkeret al., 2003a,b).

Containers, pumps or tanks used for holding or transporting unprocessed raw materials, such as raw meat and poultry or unpasteurized liquid egg, occasionally have subsequently been used for processed products without any cleaning (Morgan et al., 1993; Evanset al., 1996; Hennessyet al., 1996; Llewellynet al., 1998). This certainly represents a major deviation from good hygienic practices. Recontamination has also occurred as the consequence of ineffective or inadequate cleaning and disinfection. The poor hygienic design of equipment is then often the cause of these problems. Attention must be paid to a correct design of equipment and helpful recommendations in this respect as well as guidelines for the validation of equipment cleanability have been published (EHEDG, 1997).

Publications describe the impact of, for example, aerosols from drains on the dissemination of L.monocytogenes in fish processing facilities (Rorvik et al., 1997), from drip trays, cooling units (Goff and Slade, 1990; Anonymous, 1999) or through

hosing under high pressure. The probability of contamination has been modeled by different authors and either linear or quadratic relationships between the number of microorganisms in the product and the air have been proposed (Radmore et al., 1988). Dripping and splashing is of course also an important mode of spreading microorganisms and the mathematical distribution of microorganisms from falling drops has recently been modeled (Pielaat, 2000).

Transfer of pathogens by food handlers, in particular from hands, is of particular importance in the home and in food service establishments (Chenet al., 2001; Montville et al., 2001; Bloomfield, 2003). Many publications identified poor personnel hygiene. Deficient or absence of hand washing has in particular been identified as the causative mode of transmission of the pathogens. Hands are considered very important in the transfer of organisms with low infectious doses such asSalmonella and Shigella, viruses and pathogenic Escherichia coli (Snyder, 1998). Quantifying the cross-contamination risk associated with various steps in the food preparation process can provide a scientific basis for risk management efforts in both home and food service kitchens. However, if the product and the storage conditions support growth of micro-organisms, then this mode of transmission and recontamination may become important for other pathogens as well.

During handling and preparation, bacteria are transferred from contaminated hands of food handlers to food and subsequently to food contact surfaces (Montvilleet al 2002). Poor hygiene particularly deficient or absence of hand-washing has been identified as the causative mode of transmission (Reijet al. 2003). Proper hand washing and disinfection has been recognized as one of the most effective measures to control the spread of pathogens, especially when considered along with the restriction of ill workers (Adler 1999, Montvilleet al. 2001).

Pests such as insects, birds and rodents have been recognized as important carriers of pathogens and other microorganisms (Olsen and Hammack, 2000; Urban and Broce, 2000). In one interesting case a Salmonella outbreak has been traced back to amphibians, which had accidentally entered the production facility (Parish, 1998). While massive direct recontamination can be excluded, sporadic cases may be attributed to these vectors. More important, however, is the transport and ingress of pathogens into food processing environments and their possible establishment in suitable niches. Pest

management is therefore an essential preventive measure and guidelines have been published (Marriott, 1997).

Food preparation surfaces in turn become contaminated by already contaminated raw materials, contaminated hands of personnel preparing the food, rodents or insects acting as vectors (Chenet al 2001). Pether and Gilbert (1971) and Scott and Bloomfield (1990) reported that various bacteria, includingEscherichia coli, Staphylococcus aureus, andSalmonella spp., survive on hands, cloths, and utensils for hours or days after initial contact with the microorganisms, this in turn most probably contaminate the food preparation surfaces.

Water is also a vehicle for transmission of many agents of diseases and continues to cause significant outbreaks of diseases in developed and developing countries worldwide (Kirbyet al 2003). In Canada, an outbreak of E.coli was reported (Kondro 2000) due to consumption of contaminated water. It is therefore important that potable water is used throughout the production process, for cleaning equipment, or washing food, and preparing food.

Several studies previously carried out indicate that dish cloths or tea towels are vectors of unwanted microbial contaminations. Most of the organisms isolated were of food-borne concern includingSalmonella. Enriquezet al. (1997) isolated and identified 23 different bacterial species from 140 sponges, and 13 bacterial species from 56 dishcloths from US homes. The most common bacteria were Enterobacteriacae and Pseudomonas spp. Salmonella species were identified in 15% of sponges and 14% of dish-cloths.

Salmonellae occur on foods: fruits and vegetables, poultry carcasses, in poultry processing plants, and in poultry products (van Nierop et al., 2005). Poultry products are regarded as the primary vehicles of Salmonella transmission (Geornaras and von Holy, 2000). The birds that are brought to processing factories are a major source ofSalmonella and Campylobacter as they occur on the feathers, skin and in the alimentary tract of the birds. The birds may have acquired Salmonellae from contaminated feed, from environmental sources such as wild animals, rodents, birds etc. or through transmission from parent to progeny in hatcheries (Bremner and Johnston, 1996). Contaminated water sources by feaces of animals and humans carrying the bacteria are also a common method of transmitting the pathogen.

Thus by far it can be seen that there are many ways to present to all how, where, who, and why the microorganisms occur.

2.4 Ready-to-eat foods and outlets

Microbiological hazards continue to be one of the biggest threats to food safety. Ready-to-eat foods can be a major health risk for causing food diseases, especially in developing countries since the hygienic aspects of processing and vending operations are major source of concern for food control. Examples are running water, personal hygiene and protection from flies (El-Shenawy and El-Shenawy 2001). Ready-to-eat is defined as the status of the food being ready for immediate consumption at the point of sale. It could be raw or cooked, hot or chilled, and can be consumed without further heat-treatment including re-heating (Food and Environmental Hygiene Department 2002).

Some RTE foods include cooked chicken, beef, fish, goat, milk, fruits, fruit juices, and soft drinks and vegetables. All the foods sold on the street commonly known as street foods also belong to RTE foods. Street foods are usually sold on road sides in make-shift stalls, or on push-carts. RTE food outlets comprise of factories that produce foods intended to be consumed upon purchase like snacks, soft drinks, and candies; restaurants in hotels to small kiosks and stalls and semi-completed buildings that serve cooked food ready to eat upon purchase.

3. METHODOLOGY

3.1 Time and place of study

The research study was conducted in Darmaga, Bogor. Samples were collected from three ready-to-eat (RTE) food outlets. The research was carried out from January to March 2009 in the SEAFAST (South East-Asia Food and Agriculture Studies) laboratory, Bogor Agricultural University.

3.2 Types of samples

The samples analyzed were utensils washing water, tea towels, food preparation tables, and hand of food handlers . All samples were collected randomly.

3.3 Sampling plan

Sampling was repeated three times for all four samples from the three ready-to-eat food outlets. This gave a total of 36 samples being analyzed.

That is, 3 repeats x 4 types of samples x 3 food outlets = 36 samples. Statistical analysis to see if there were significant differences amongst the three RTE food outlets; Completely Randomized Design was used.

3.4 Criteria for selecting the ready-to-eat food outlets

Two criteria were used to select the food outlets. The first criterion was based on size of the outlet in terms of how many people it would be able to cater for when full. Three different sizes were selected: the smallest having a capacity to hold about 6 to 8 people; medium size about 12 to 14 people; and largest size having to more than 20 people. The layout of each RTE food outlets are given in Figure 1 of Appendix.

The second criterion was based on the type of foods sold in each outlet. Outlet I and outlet II sold many different types of RTE foods including chicken, beef, fish, sausage, egg & egg products, vegetables, meat ball & noodles (mie bakso) all served with rice, however the difference was the sales of bread/buns and cakes and other pastries, and the drink varieties and frozen goods by outlet I. Outlet II sold the same variety of cooked foods but it sold sweetened or unsweetened tea prepared upon order as well as boiled water provided I jugs on the tables. Unlike outlets I and II, outlet III sold

(chicken) or soto daging (beef). Both forms served with santen (coconut cream) or bening (clear/plain). Soto os a soup type of dish where pieces of chicken or meat are put in liquid containing coconut cream or water with added flavor from selected spices, sauces, and vegetables. Outlet served sweetened/unsweetened tea upon order and also sold fruit and jelly mixture locally calledes campur.

3.5 Sampling method

3.5.1 Collecting sample

Samples were collected using sterile glass bottles for utensils washing water samples, and sterile swabs for collecting tea towels, food preparation tables and hands of food handlers samples. Collecting samples took less than 1 hour each day as the RTE food outlets were located within walking distance from the laboratory where analysis were carried out. All sterile items, including sterile Lactose broth media forSalmonella were put in two clean and sterile ice boxes (eskies).

Figure 4. Ice boxes for sampling.

All sampling were done aseptically and done during lunch hours from 11:00am to 13:00pm when the RTE food outlets were seen to be at their busiest moments.

3.5.2 Swabbing technique

Swabbing was done to about 100cm2 surface area of the food contact surfaces (food preparation tables, tea towels and hands of food handlers) using moist cotton swabs. According to AS 4696; 2007, swabbing of greater than 100cm2 would get the swabs overloaded, therefore swab are recommended to sample area sizes 100cm2.

3.5.1.1 Utensils’ washing water

Water samples were collected aseptically in sterile glass jars with lids according to Harrigan and McCance (1976). Collecting water samples was done quite differently for outlet III to outlets I and II. Both outlets I and II had washing troughs/buckets keeping the washing water whilst in outlet III, running water was used.

The manner in which the water samples were collected was influenced by how the washing water was kept. In outlets I and II, the sterile glass jars were dipped into the water after the water was stirred by shaking the trough/bucket, ensuring that hand of the sampler didn t touch the water to avoid contamination. Means of using a container to fetch the water to fill the jars was also decided against with the same reason to avoiding contamination. In outlet III, running tap water was used therefore the glass jar was held below the running tap to collect the water sample.

3.5.1.2 Tea towels

Sampling of tea towels is usually done by rinsing technique. In this research, the tea towels were placed on sanitized and alcohol sterilized flat surface and moist cotton swabs were used to collect microbial samples from top side of the tea towels. The cotton swabs after covering the required surface area (10cm x 10cm) were then broken off from the tail and placed into 10mls of diluents containing sterile bottles, for coliform samples. For Salmonella samples, the broken off swab heads were placed into 225ml lactose broth media.

The same tea towels were sampled for the three repeats from the three RTE food outlets. Swabs of 1.0 1.5cm thick and 3.5 4.0cm length were used to take the sample from one side of the tea towel.

Figure 5. Diagram for swabbing action.

3.5.1.3 Food preparation tables

Food preparation tables sampling was done by swabbing technique using moist cotton swabs of 1.0 1.5cm thick and 3.5 4.0cm length of a defined area of 10cm x 10cm for all three RTE food outlets. All the used swabs were carefully placed into 10ml of diluent contained in sterile bottles after having the handles broken off (for coliforms);

forSalmonella samples, the broken off swab heads were placed into 225ml lactose broth media.

3.5.1.4 Hands of food handlers

All the hands were swabbed by rubbing the swab over the palms and between the fingers of the hands of the workers. According to HyServe (2009), a company specialized in producing an ATP surface hygiene detector stated that typical inspection points and swabbing manner for hands and fingers were swabbing of the palm, between fingers, and cracks of nails. This enabled in this research the method of swabbing used for sampling hands of food handlers.

Size of the hands of all three workers in each outlet may have been of different size but were not exactly determined because of the way swabbing was done (over palm and between fingers). However, general assumption is that female hands are smaller compared to male hands and so according to this; it was assumed that since in outlets I and III, female hands were sampled thereby would have smaller hands to the male personnel in outlet II. Thus, estimation of the surface area of the hands and fingers were determined by drawing a sketch diagram around the hands sampled and measurements estimated. The area was estimated by measuring the palm width and length, and individual finger width and lengths and adding all the areas together (area of palm + area of finger 1 + area of finger 2 + area of finger 3 + area of finger 4 + area of finger 5). Swab samples of hands covered area size slightly higher than 100cm2.

After swabbing the swabs after breaking off the handles, were placed in diluent containing bottles (for coliforms) and into 225ml of Lactose media (forSalmonella).

3.6. Isolating and identifyingSalmonella species

The USFDA/CFSAN (2007), (United States Food and Drug Administration/Center for Safety and Nutrition) bacterial analytical methods (BAM) of detecting Salmonella was used. This conventional method takes up to 6 days. It is similar to the other conventional methods of detection except the use of media employed and slight difference in incubation temperature which is based on the type of media used. Figure 4 shows the flow diagram of the steps undertaken in the analysis of Salmonella species.

Figure 6. Diagram of the procedure for detection ofSalmonella sp.

3.6.1 Non-selective enrichment (resuscitation)

Swabs were placed into 225 ml lactose broths in Erlenmeyer flasks with lids; the contents were swirled to loosen the bacterial cells from the cotton swabs of the flask and let stand for 60 ± 5 minutes at room temperature. The lactose broth and swab sample mixtures were then incubated at 35°C for 24 ± 2 hours. Swab samples were for tea towels food preparation tables and hands of food handlers. For the utensils washing water 25g of the samples were weighed out and added to 225 ml of lactose in sterile capped Erlenmeyer flasks (500 ml), followed by incubation at 35°C for 24 ± 2 hours.

25g of each swab isolate samples; 25g of water sample

225ml of lactose broth; mix for 2 minutes (Let stand for 60±2minutes)

Incubate: 24±2 hours at 35°C.

0.1ml of culture into 10ml RV medium Incubate: 42 ± 0.2°C for 24 ± 2 hours

1ml of culture into 10ml TT broth Incubate: 43 ± 0.2°C for 24 ± 2 hours

XLD, HE, BS agar media 35 °C for 24 ± 2 hours.

Inspect plates for the presence of characteristic colonies and any primary bochemical reactions

1) Confirmation of suspected colonies

2) Purify suspected colonies in Nutrient Agar; incubation at 35°C for 24 ± 2 hours

Biochemical confirmation Serology using antisera. - Agglutination reaction.

3.6.2 Selective enrichment

From the non-selective enrichment sample mixture, 0.1 ml was transferred to 10 ml of Rappaport-Vassiliadis (RV) medium, and another 1 ml mixture to 10 ml of tetrathionate (TT) broth. The tube mixture compositions were then shaken vigorously by Vortex. RV medium were incubated at 42 ± 0.2°C for 24 ± 2 hours in circulating, thermostatically-controlled water bath. TT broths were incubated at 43 ± 0.2°C for the same time frame.

3.6.3 Plating on selective solid media

The RV and TT broth mixtures after the period of incubation were then shaken/mixed (vortex) and streaked onto Bismuth sulfite (BS) agar, xylose lysine desoxycholate (XLD) agar, and Hektoen enteric (HE) agar with 3 mm loopfuls (10 µl). The streaked plates then were incubated at 35 °C for 24 ± 2 hours.Note: BS plates were prepared a day before streaking, and stored in dark at room temperature until streaked.

Typical salmonella colonies from the plating would be as follows: 1) Hektoen Enteric (HE) agar indication ofSalmonella being present be denoted by blue-green to blue colonies with or without black centers. Many cultures ofSalmonella produce large, glossy black centers or appear as almost completely black colonies; 2) Xylose lysine desoxycholate (XLD) agar would be denoted by pink colonies with or without black centres; 3) Bismuth sulfite (BS) agar Brown, gray or black colonies sometimes with metallic sheen would be an indication ofSalmonella. The surrounding usually is brown at first, but may turn black in time with increased incubation, producing the so-called halo effect.

The atypicalSalmonella colony morphology would be as follows: 1) HE and XLD agars would produce yellow colonies with or without black centers; 2) BS agar some of the strains would show green colonies with little or no darkening of the surrounding medium.

3.6.4 Sub-culturing of presumptiveSalmonella

Typical Salmonella colonies from BS agar, HE agar, and XLD were then transferred into triple sugar iron agar slants (TSI) and lysine iron agar slants (LIA) using sterile streak and butt stab needle. The streaked and butt stabbed TSI and LIA slants

Table 9. Grading criteria using the modified Badan POM criteria.

Total deficiencies Rating

Minor Major Serious Critical Frequency of audit Grading Grade definition

I 0 - 10 0 - 5 0 0 once/6 months A Very Good

II >11 11 - 20 1 - 10 0 once/4 months B Good

III N/A >20 10 - 20 0 once/2 months C Fair

IV N/A N/A >21 >2 once/1 month D Poor

N/A=Not applicable

Photos of the ready-to-eat food outlets

Figure 1. Pictures of the ready-to-eat food outlet I.

Cakes and other pastries Variety of processed drinks

Variety of cooked foods Eating area

Storage/preparation area Utensil washing area

Figure 2. Pictures of the ready-to-eat food outlet II.

Variety of cooked foods Entrance of food outlet

Food preparation area Utensil washing area

Eating area

8.3 Coliform counts using 3-tube MPN

Table 10. MPN index and 95% confidence limits for various combinations of positive tubes in a 3 tube dilution series using inoculums quantities of 1, 0.1, and 0.01 g (ml).

Positive tubes Conf. lim. Positive tubes Conf. lim. 1.0 0.1 0.01

MPN/g

Low High 1.0 0.1 0.01

MPN/g

Low High 0 0 0 <0.3 -- 9.5 2 2 0 2.1 4.5 42 0 0 1 0.3 0.15 9.6 2 2 1 2.8 8.7 94 0 1 0 0.3 0.15 11 2 2 2 3.5 8.7 94 0 1 1 0.61 1.2 18 2 3 0 2.9 8.7 94 0 2 0 0.62 1.2 18 2 3 1 3.6 8.7 94 0 3 0 0.94 3.6 38 3 0 0 2.3 4.6 94 1 0 0 0.36 0.17 18 3 0 1 3.8 8.7 110 1 0 1 0.72 1.3 18 3 0 2 6.4 17 180 1 0 2 1.1 3.6 38 3 1 0 4.3 9 180 1 1 0 0.74 1.3 20 3 1 1 7.5 17 200 1 1 1 1.1 3.6 38 3 1 2 12.0 37 420 1 2 0 1.1 3.6 42 3 1 3 16.0 40 420 1 2 1 1.5 4.5 42 3 2 0 9.3 18 420 1 3 0 1.6 4.5 42 3 2 1 15.0 37 420 2 0 0 0.92 1.4 38 3 2 2 21.0 40 430 2 0 1 1.4 3.6 42 3 2 3 29.0 90 1,000 2 0 2 2.0 4.5 42 3 3 0 24.0 42 1,000 2 1 0 1.5 3.7 42 3 3 1 46.0 90 2,000 2 1 1 2.0 4.5 42 3 3 2 110 180 4,100 2 1 2 2.7 8.7 94 3 3 3 >110 420

Table 11. For 3 tubes each at 0.1, 0.01, and 0.001 g inocula, the MPNs per gram and 95 percent confidence intervals.

Positive tubes Conf. lim. Positive tubes Conf. lim.

0.10 0.01 0.001

MPN/g

Low High 0.10 0.01 0.001

MPN/g

Low High 0 0 0 <3.0 -- 9.5 2 2 0 21 4.5 42 0 0 1 3.0 0.15 9.6 2 2 1 28 8.7 94 0 1 0 3.0 0.15 11 2 2 2 35 8.7 94

0 1 1 6.1 1.2 18 2 3 0 29 8.7 94

0 2 0 6.2 1.2 18 2 3 1 36 8.7 94

0 3 0 9.4 3.6 38 3 0 0 23 4.6 94

1 0 0 3.6 0.17 18 3 0 1 38 8.7 110

1 0 1 7.2 1.3 18 3 0 2 64 17 180

1 0 2 11 3.6 38 3 1 0 43 9 180

1 1 0 7.4 1.3 20 3 1 1 75 17 200

1 1 1 11 3.6 38 3 1 2 120 37 420

1 2 0 11 3.6 42 3 1 3 160 40 420

1 2 1 15 4.5 42 3 2 0 93 18 420

1 3 0 16 4.5 42 3 2 1 150 37 420

2 0 0 9.2 1.4 38 3 2 2 210 40 430

2 0 1 14 3.6 42 3 2 3 290 90 1,000 2 0 2 20 4.5 42 3 3 0 240 42 1,000 2 1 0 15 3.7 42 3 3 1 460 90 2,000 2 1 1 20 4.5 42 3 3 2 1100 180 4,100 2 1 2 27 8.7 94 3 3 3 >1100 420 --Source: USFDA/CFSAN 2003.

Table 12. Least Significant difference between RTE food outlets for total coliforms. Difference of

Averages BNT, t0.025 BNT, t0.05 Comments

I - II 3636.00 3915.82 3113.40 Not significant at t0.025, significant at t0.05 I - III 4430.00 3915.82 3113.40 Significant at both t0.025 and t0.05 Water

II - III 794.00 3915.82 3113.40 Not significant

I - II 556.67 333.21 264.93 Significant at both t0.025 and t0.05 I - III 556.67 333.21 264.93 Significant at both t0.025 and t0.05 Tea towel

II - III 382.33 333.21 264.93 Significant at both t0.025 and t0.05

I - II 0.00 245.38 195.10 Not significant

I - III 426.67 245.38 195.10 Significant at both t0.025 and t0.05 Food prep. tables

II - III 426.67 245.38 195.10 Significant at both t0.025 and t0.05

I - II 245.13 108.36 86.16 Significant at both t0.025 and t0.05 253.33 108.36 86.16 Significant at both t0.025 and t0.05 Hands of food

handlers

II - III 8.20 108.36 86.16 Not significant

Table 13. Least Significant difference between RTE food outlets for fecal coliforms. Difference of

Averages BNT, t0.025 BNT, t0.05 Comments

I - II 24.00 42.91 34.12 Not significant

I - III 40.67 42.91 34.12 Not significant at t0.025, significant at t0.05 Water

II - III 16.67 42.91 34.12 Not significant

I - II -1.80 21.26 16.91 Not significant I - III 1.33 21.26 16.91 Not significant Tea towel

II - III 3.13 21.26 16.91 Not significant

I - II 39.00 26.89 21.38 Significant at both t0.025 and t0.05 I - III 45.93 26.89 21.38 Significant at both t0.025 and t0.05 Food prep. tables

II - III 6.93 26.89 21.38 Not significant

I - II 1.67 1.48 1.17 Significant at both t0.025 and t0.05

1.87 1.48 1.17 Significant at both t0.025 and t0.05

Hands of food handlers

8.4 List of Equipment and Materials

1. Sterile, 16 oz (500 ml) wide-mouth, screw-cap jars, sterile 500 ml Erlenmeyer flasks, sterile 250 ml beakers, sterile glass or paper funnels of appropriate size, and, optionally, containers of appropriate capacity to accommodate composited samples

2. Sterile, bent glass or plastic spreader rods

3. Balance, with weights; 2000 g capacity, sensitivity of 0.1 g 4. Balance, with weights; 120 g capacity, sensitivity of 5 mg 5. Incubator, 35 ± 2 °C

6. Water bath, 49 ± 1°C

7. Water bath, circulating, thermostatically-controlled, 43 ± 0.2°C 8. Water bath, circulating, thermostatically-controlled,42 ± 0.2°C

9. Sterile spoons or other appropriate instruments for transferring food samples 10. Sterile culture dishes, 15 x 100 mm, glass or plastic

11. Sterile pipets, 1 ml, with 0.01 ml graduations; 5 and 10 ml, with 0.1 ml graduations

12. Inoculating needle and inoculating loop (about 3 mm id or 10 5l), nichrome, platinum-iridium, chromel wire, or sterile plastic

13. Sterile test or culture tubes, 16 x 150 mm and 20 x 150 mm; serological tubes, 10 x 75 mm or 13 x 100 mm

14. Test or culture tube racks 15. Vortex mixer

16. Fisher or Bunsen burner

17. pH test paper (pH range 6-8) with maximum graduations of 0.4 pH units per color change

18. Plastic beakers, 4 liter, autoclavable, for holding plastic bag during shaking and incubation.