An-Najah National University Faculty of Graduate Studies Assessment of Microbial Quality of Food Samples in Nablus District By Omayya Morshed Mohammad Hammad Supervisors Dr.Yahya R. Faidy Dr. Issam A. Al –Khatib Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Public Health, Faculty of Graduate Studies, at An-Najah National University, Nablus, Palestine. 2004 3 Dedication To MY MOTHER AND FATHER WITH LOVE AND GRATITUDE. MY HUSBAND WITH LOVE, AND DEEP APPRECIATION. MY SISTER AND BROTHERS AND MY BELOVED CHILDREN KHALED, TAREQU, RAHAF, AND MOHAMMAD. 4 ACKNOWLEDGMENTS I would like to express my special thanks and gratitude to my supervisor, Dr. Y. Faydi for his supervision, encouragement, and help throughout this project. It is a pleasure to record here my deepest gratitude to Dr. Issam A. Al –Khatib for his keen observations, stimulating suggestions, invaluable insights and guidance. Thanks are due to the staff of the Environmental Health Department in Nablus Health Directorate/Ministry of Health. My deepest gratitude to my beloved husband who gave me leisure and freedom to complete this thesis. His love, encouragement, help and advice were invaluable. 5 TABLE OF CONTENTS Subject Page Committee Decision ii Dedication iii Acknowledgments iv Contents v List of Tables viii Abbreviations ix Abstract x CHAPTER ONE: INTRODUCTION 1 1.1 General information about Nablus district. 2 1.1.1 Location 2 1.1.2 Topography 2 1.1.3 Geographical location. 3 1.1.4 Temperature. 3 1.1.5 Humidity 3 1.1.6 Population 6 1.2 Statement of the problem 6 1.3 Objectives 6 1.4 Limitations 7 CHAPTER TWO: LITERATURE REVIEW 8 2.1 Definition of terms 9 2.2 Main foodborne diseases 10 2.2.1 Bacterial infections 10 2.2.2 Viral infections 23 2.2.3 Yeast 26 2.2.4 Moulds 26 2.3 Factors affecting the growth of microorganisms in food. 26 2.4 Food microbial quality indicators 31 2.5 Factors contributing to the emergence of foodborne illness 32 2.6 Important definitions that are of great concern 35 2.7 Principles of handling and hygienic control of foods in restaurants and home. 36 6 Subject Page 2.8 General rules to reduce the hazards of foodborne in restaurants and food preparation services. 38 CHAPTER THREE: METHODOLOGY 42 3.1 Sample collection 43 3.2 Receiving samples at the laboratory 43 3.3 Samples analysis 44 3.4 Acceptance of samples 45 3.5 Methods of detections of microorganisms in food samples at lab. 46 CHAPTER FOUR: RESULTS 50 4.1 Distribution of food samples 50 4.2 Microbial quality and food types 54 4.3 Microbial quality and food source 57 4.4 Microbial quality and food and cooked foods 60 4.5 Microbial quality and food preservatives 62 4.6 Numbers and percentages of acceptable and un acceptable samples during the different months of the years 1995- 2003. 70 4.7 Numbers, percentages of acceptable, and un acceptable samples during the years 1995-2003. 68 4.8 4.8 Numbers and percentages of acceptable and un acceptable samples during the four seasons for the years 1995-2003. 70 CHAPTER FIVE :DISCUSSION 75 5.1 Food types and acceptance of various microbiological tests. 75 5.1.1 Fruits and vegetables. 75 5.1.2 Salads. 76 5.1.3 Meats and diary products. 77 5.2 Acceptance of samples source and various microbiological tests. 79 5.2.1 Restaurants. 79 5.2.2 Food factories. 80 5.3 Acceptance of cooked samples and various microbiolo- gical tests. 81 7 Subject Page 5.4 Acceptance and distributions of food samples by month, seasons, years. 82 5.4.1 Distribution of samples during studied years. 82 5.4.2 Distribution of samples through the different seasons. 82 5.4.3 Distribution of samples through the different months. 83 Conclusion 84 Recommendations 85 References 86 Appendix 92 Abstract in Arabic ب 8 LIST OF TABLES Subject Page Table 1. The maximum, minimum, and average temperatures and percentages of humidity in Nablus district 1997- 2003. 4 Table 2. Characteristics of some microbiological pathogens that have been linked to outbreaks of food illness 25 Table 3. Foodborne illnesses reports from restaurants in USA 1996 37 Table 4. Sample types and acceptance of various microbiolo- gical tests. 52 Table 5. Sample sources and acceptance of various microb- iological tests. 55 Table 6. Numbers and percentages of acceptable and unacc- eptable for cooked samples 58 Table 7. Numbers and percentages of acceptable and un- acceptable for samples with or without presserv- atives. 61 Table 8. Numbers and percentages of acceptable and unacceptable samples during the twelve months of the years 1995-2003. 63 Table 9. Numbers and percentages of acceptable and unacceptable samples during the years 1995-2003. 68 Table 10. Numbers and percentages of acceptable and unacceptable samples during the four seasons for the years 1995-2003 71 9 Abbreviations ARIJ: Applied Research Institution of Jerusalem aw: Water Activity or water availability. CDC: Centers for Disease Control and Prevention. CFSAN: Centers for Food Safety and Applied Nutrition. CFU: Colony Forming Unit. EHEC: Entero Hemorrhagic Escherichia Coli. E.Coli: Escherichia Coli FDA: Food and Drug Administration. FC: Faecal Coliform. HACCP: Hazard Analysis and Critical Control Points. HAV: Hepatitis A virus. NIAID: National Institute of Allergy and Infectious Disease. P - value: Probability (Significant Level) SA: Staphyllococcus Aureus. Sal: Salmonella. TAC: Total Aerobic Count. TC: Total Coliform. 10 Assessment of Microbial Quality of Food Samples in Nablus District By Omayya Morshed Mohammad Hammad Supervisors Dr.Yahya R. Faidy Dr. Issam A. Al –Khatib ABSTRACT Data of microbiological food examination recorded between 1995- 2003 at Environmental Health Department of Ministry of Health were analyzed and studied for microbial contamination in order to assess the variations of bacterial quality of food by type, source, months, seasons, years. A total of 1052 samples were collected from Environmental Health Department of Ministry of Health for routine test of microbiological quality for public health issue by Environmental Health Inspectors of Nablus district during the period 1995-2003. This study showed that salads, meats and chickens and diary products had the highest percentages of unaccepted samples tested for TAC: these percentages are (62.1%), (14%) and (5.6%) respectively. This study showed that the restaurants had the highest percentages of unacceptable samples tested for TAC, TC, S aureus, Yeast and Moulds These percentages are (56.9%), (65.6%), (1%), (75.9%), and (3%) respectively . This study conclude that lack of monitoring in restaurants, and food factories, and unsystematic food sampling system, due to the current situation such as closure and separation of the Palestinian territories by military checkpoints has created a lot of difficulties facing reporting, food sampling and consequently food control. 11 The study recommends conducting health education programs about food safety to food handlers, holding educational programs for public, and increasing the numbers of trained food inspectors. 12 CHAPTER ONE 13 CHAPTER ONE Introduction 1.1 General Information about Nablus District 1.1.1 Location Nablus district is located in the northern part of the West Bank. It is bounded by Jenin from the north, Tulkarm from the west, Ramallah and Jericho from the south and the Jordan River from the east. The district is located between 349 m below sea level and 918 m above sea level, (Applied Research Institute Jerusalem (ARIJ), 1996). 1.1.2 Topography The topography of Nablus district can be divided into four parts: 1- Jordan Valley: is located between Jordan River and the eastern slopes with elevation ranges between 349 m below sea level to 100 m above sea level. 2- The eastern slopes: are located between Jordan valley and the mountains. They are characterized by steep slope which contribute to forming young wadis such wadi El Badan. 3- Mountain crests: from the water shed line and separate the eastern and western slopes. Elevation ranges on average between 750 and 800 meters above sea level. 4- Western slopes, characterized by gent slopes, with elevation ranges between 250-500 meters above sea level, (ARIJ, 1996). 14 The highest point in the district reaches 918 m above the sea level at Jabal Ibal, while the lowest elevation is 349 m below sea level at the south east corner of the district, (Nablus municipality, 2002). 1.1.3 Geographical Location Nablus district is located at the northern latitude earth grid 32:12 and the eastern latitude earth grid 35:16. It's about 110 km far away from Amman, 42 km from Mediterranean, 66km from Jerusalem and Jenin and it is 550 m high from sea level (Nablus Municipality, 2003). 1.1.4 Temperature The geographical position of Nablus district in the northern part of the West Bank gives it a comparatively lower temperature range than the other districts. During January, the coldest month, the average maximum temperature reaches 19.9 Cº, and the average minimum temperature reaches 2.6 Cº. During August, the hottest month, the average maximum temperature is 33.8 Cº and the average minimum temperature is 19.2Cº, as shown in table (1) (Palestinian Meteorology Department, (PMD) 2004). 1.1.5 Humidity The mean annual relative humidity of Nablus district is 62%. During the khamaseen period, the relative humidity decreases to reach its minimum value of 18.0 % (in May). Maximum humidity of 89.5%, 90.5%, 91.0% are usually registered in December, January, and February respectively. This value increases gradually at night (ARI, 1996 and PMD, 2004). 15 Table 1. Maximum, minimum and average temperature degrees and humidity percentages for Nablus district for the years 1997- 2003 Month Year/ Temperature 1997 1998 1999 2000 2001 2003 January Max 24.0 17.0 19.8 23.5 21.0 14.0 Av. 14.3 8.5 11.6 12.25 12.4 9.0 Min. 4.6 0.0 3.4 0.0 3.8 4.0 February Max 22.5 20.0 22.2 17.9 25.0 13.0 Av. 11.25 11 13.1 10.8 14.7 8.6 Min. 0.0 2.0 4.0 3.8 4.5 4.2 March Max 23.5 24.6 26.2 25.5 32.2 14.2 Av. 12.8 13.3 15.9 14.2 20.1 10.8 Min. 2.2 2.0 5.6 3.0 8.0 7.5 April Max 35.5 36.0 31.5 33.2 32.4 21.7 Av. 18.0 21.1 19.4 21.1 20.7 15.1 Min. 0.6 6.2 7.4 9.0 9.0 8.6 May Max 35.5 36.0 37.5 33.5 37.3 34.0 Av. 22.8 32.5 24.9 22.2 24.6 24.5 Min. 10.2 11.0 12.4 11.0 12.0 15.0 June Max 36.1 33.2 32.2 35.0 35.0 33.4 Av. 25.0 23.7 24.1 25.4 25.2 24.4 Min. 14.0 14.3 16.0 15.8 15.4 15.4 July Max 33.5 34.4 32.5 37.5 33.5 35.4 Av. 26.1 26.4 25.4 28.2 26.0 27.3 Min. 18.7 18.4 18.3 19.0 18.6 19.2 August Max 31.6 37.0 35.5 32.5 33.4 32.5 Av. 24.2 28.5 26.9 26.0 26.8 26.2 Min. 16.8 20.0 18.4 19.6 20.2 20.0 September Max 24.5 37.5 37.2 36.0 31.6 32.6 Av. 24.2 27.0 27.9 25.7 24.8 24.1 Min. 14.0 16.6 18.6 15.5 18.0 15.6 October Max 35.0 34.9 30.6 33.2 33.4 33.0 Av. 23.3 23.6 22.5 21.8 22.6 22 Min. 23.3 23.6 22.5 21.8 22.6 22 November Max 11.6 12.4 14.4 10.4 11.8 11.0 Av. 17.6 19.6 17 17.4 15.6 19.6 Min. 10.0 10.8 5.0 9.4 5.8 9.6 December Max 23.0 26.5 24.2 19.5 21.8 19.5 Av. 13.8 15.5 14.4 12.7 13.3 12.1 Min. 4.6 4.5 4.6 6.0 4.8 4.8 January. Max 93 91 92 95 87 85 16 Month Year/ Temperature 1997 1998 1999 2000 2001 2003 Av. 63 70.5 66 70 60 59.5 Min. 33 50 40 45 33 34 February Max 91 92 87 93 93 90 Av. 62 67 59.5 62.5 65 61 Min. 33 42 32 32 37 32 March. Max 88 91 86 86 85 86 Av. 58.5 62.5 55.5 64.5 52.5 54 Min. 29 34 25 43 20 22 April Max 86 87 84 79 79 80 Av. 51 51 52.5 52.5 48.5 52 Min. 16 15 21 26 20 24 May Max 80 90 73 71 83 66 Av. 50 54 42.5 48.5 51.5 38.5 Min. 20 18 12 26 20 11 June Max 74 74 76 74 70 68 Av. 47.5 51.5 55 47 45.5 45.5 Min. 21 29 34 20 21 23 July Max 71 76 75 73 74 71 Av. 51 50 60.5 52.5 55.5 46.5 Min. 31 24 46 32 37 22 August Max 78 74 75 79 74 75 Av. 62 57 59 63 66 60 Min. 46 40 43 47 58 45 September Max 84 76 77 77 76 72 Av. 62 45.5 44.5 59 64.5 54 Min. 40 15 12 41 53 36 October Max 82 77 77 83 88 80 Av. 54.5 43 59.5 53 61.5 54.5 Min. 27 9 42 23 35 29 November Max 91 80 81 77 90 89 Av. 62 57 48.5 52 66.5 58.5 Min. 33 34 16 27 43 28 December Max 92 87 88 89 92 89 Av. 62.5 58.5 54.5 65.5 66 66 Min. 33 30 21 42 40 43 *The information for the year 2002 is not available. ** Max: means maximum, Min: means minimum, Av. Means average 17 1.1.6 Population The estimated total population of the Nablus District is 341,412 for the year (2004), representing 8.75% of the total population of Palestine, (Palestinian Central Bureau of Statistics (PCBS), 2004). Approximately 47.9% (120382) of the population of the Nablus district live in rural area. 10.5% (approximately 26447) lives in refugee camps and 41.6% (104563) lives in communities under municipalities' administration (PCBS, 2003). 1.2 Statement of the Problem The aim of this study is to assess microbial quality of food in Nablus district and to study the related food borne diseases, to do so, food samples were taken from different sources, different time and during the years 1995-2003 and tested for microbial contamination 1.3 Objectives The objectives of the study is to estimate the variations of bacterial quality of food by the type, source, months, seasons, years, the effect of treating food items by preservatives or heat before consumption in factories or preparation sites and recommending of some solutions to the problem of food contamination are also suggested. 1.4 Target Groups - Samples were collected by MOH – Environmental health department from different sites, times during the years 95-2003 and studied at the central public health lab (CPHL) in Rammallah. 18 - Parameters studied were TAC, TC, Salmonella, Staph. Yeast and molds, standard methods were used to collect and test samples. - The results were categorized into accepted, or unaccepted according to Palestinian standards, WHO Standards and Gulf states standards. 1.5 Limitations: 1. One of the limitations of this study is that the samples did not include the location of the samples were (city, camp, and village) because it was neglected by the sheet designer in the MOH. 2. For security purposes the source of samples was deleted by the ministry of health inspectors so we will find in results that a large number of samples of unknown source. 3. data in the years 1996, 1998 and 2001 were not available from records of environmental health department thus were not included in this study. 19 CHAPTER TWO 20 CHAPTER TWO LITRATURE REVIEW 2.1 Definition of Terms. Food Poisoning is an illness caused by ingestion of contaminated food. 1- Chemical Food poisoning is an illness caused by ingesting food containing chemical poison. 2- Food Intoxication is an illness caused by ingesting food containing microbial toxins. 3- Foodborne Diseases are infectious diseases spread through or by consuming contaminated foods or beverages (National Institute of Allergy and Infectious Disease (NIAID) 2002). Foodborne diseases are common, distressing and some times life- threatening problem for millions of people around the world. In USA, centers for disease control and prevention (CDC) "estimates 76 million people suffer foodborne illnesses each year accounting for 325,000 hospitalizations and more than 5,000 deaths", (CDC. 2002). According to WHO "while less well documented, developing countries, bear the brunt of the problem of the presence of a wide range of foodborne illnesses, including those caused by parasites, bacteria, viruses", (WHO, 2002). Foodborne outbreak is defined as illness in at least two persons with digestive symptoms that can be attributed to the same food source, (Hopkins, 2000). 21 Small outbreaks are likely to occur more frequently than outbreaks involving a large number of persons. Such outbreaks would be expected to be especially prolific in families or among other groups of people living together because they would often be exposed to the same risk factors (Ethelberg and Olsen, 2003). 2.2 Main Food borne Diseases 2.2.1- Bacterial Infections The Majority of all Cases of Food Poisoning are due to Bacterial Infections. 2.2.1.1- Salmonellosis In 1885 An American veterinary scientist, Daniel E.Salmon, discovered the first strain of Salmonella from the intestine of pig. This strain was called Salmonella Choleraesuis, the designation that is still used to describe the genus and species. Salmonella is type of bacteria that cause typhoid fever and many other infections of intestinal origin (Clark, 2002). Salmonella species are Gram-negative, flagellated facultative anaerobic bacilli most are motile with peritrichom flagella, ferment glucose with the production of acid and gas or acid only .Some Salmonella produce H2S (Jawetz, Melnick, and Adelberg's, 2004) Compared with other gram-negative rods, Salmonella is relatively resistant to various environmental factors, grow at temperatures between 8Cº and 45Cº and in a pH range of 4 to 8 (Adams, 1995). Salmonella is often pathogenic to humans and animals .Infection results from the ingestion of food or water containing sufficient number of these bacteria to reach and invade the small intestine (Adams, 1995). 22 Salmonella produce three main types of disease in humans: Enteric fever (Typhoid fever), Bacteremia and Enterocolitis, but mixed forms are frequent (Jawetz, Melnick, and Adelberg's, 2004). Most cases of salmonellosis are considered to be endemic or sporadic because they are not clustered. The usual explanation for endemic cases is the inappropriate handling in kitchens and restaurants of contaminated food (including improper storage, undercooking, or cross contamination) (Blaser, 2004). According to CDC "Every year, approximately 40,000 cases of Salmonellosis are reported in the United States and it is estimated that approximately 600 persons die each year with acute Salmonellosis (CDC, 2004). According to Hopkins "In France, Salmonella is involved in 75.6 percent of reported foodborne outbreaks with an identified causative organism, 70 percent of which occur in family household" (Hopkins, 2000). Symptoms of salmonellosis in clued fever, abdominal pain, diarrhea and vomiting. Associated Foods 1- Contaminated beef meats, raw poultry. 2- Unwashed fruits. 3- Vegetables grown in contaminated soils. 4- Eggs (NIAID, 2002). 23 Some types of Salmonella can infect a hen's ovary so that the internal contents of a normal looking egg can be contaminated with Salmonella even before the shell is formed (CDC, 2003). The consumption of under cooked eggs or egg products contaminated with S. enteritidis is the major cause of salmonellosis (Hennessy, 2004). In USA, Salmonella enteritis has become the most commonly reported serotype of Salmonella causing disease in humans (Hennessy, 2004). 2.2.1.2 Typhoid Fever Typhoid fever is a life threatening illness caused by Salmonella typhi. (CDC, 2001). Salmonella Typhi lives only in humans; persons with typhoid fever carry the bacterium in their blood stream and intestinal tract (CDC, 2001). Typhoid fever is common in most parts of the world except in industrialized regions such as United States, Canada, Western Europe, Australia and Japan (CDC, 2001). According to WHO "Typhoid fever is still common in the developing world, where it a affects about 12.5 million persons each year, (WHO, 2004). Patients with typhoid fever usually have a sustained fever as high as (39-40C ْ◌). They may also feel weak or have stomach pain, headache or loss of appetite. In some cases, patients have a rash of flat and rose- colored spots. The only way to know for sure if an illness is typhoid fever is to have samples of stool or blood tested for the presence of S.typhi (CDC, 2001). 24 The disease is Prevented by: 1. Avoid foods and beverages from street vendors. 2. Avoid unpasteurized milk and milk products. 3. Cook poultry and egg thoroughly. 4. Avoid unwashed and unpeeled fruits. 2.2.1.3 Escherichia Coli Infections ( Facal Coliform) E.coli is facultative gram negative rods, motile with beritrichom flagella organism: it causes traveler's food poisoning. Certain types of E.coli can cause food borne illness. Harmless strains of E.coli is found widely in nature, most strains are considered to be part of GIT of man and other warm blood animals including intestinal tracts of humans and warm- blooded animals (NIAID, 2002). Several different strains of harmful E.coli can cause diarrheal disease. A particularly dangerous type is called entero hemorrhagic E.coli of EHEC. Often causes bloody diarrhea and can lead to kidney failure in children or people with weakened immune systems (FDA, 2001). In 1982 scientists identified the first dangerous strain in the United States, the type of harmful E.coli most commonly found in USA named 0157:H7 which refers to chemical compounds found on the bacterium's surface. This type produces one or more related, powerful toxins which can severely damage the lining of the intestines (FDA, 2001). According to CDC "an estimated 73,000 cases of infection and 61 deaths occur in the United States each year" (CDC, 2004). 25 In USA, 47 airline passengers suffered from illness strongly associated with eating garden salad made from iceberg and romaine lettuce, endive, and shredded carrots (Beuchat, 1996). In another outbreak, 78 lodge guests become ill after consuming tossed salad as part of a buffet dinner. The salad contained several ingredients, including onions, carrots, peppers, mushrooms and tomatoes, (Beuchat, 1996). In 1996, an outbreak of E.Coli 0157: H7 in Japan affected over 6.300 school children resulted in 2 deaths. This is the largest out break ever recoded for this pathogen (WHO, 2002). Associated Food 1. Undercooked or row hamburgers or ground beef. 2. Unpasteurized milk, apple juice, and apple cider. 3. Unwashed and contaminated raw vegetables and fruits (NIAID, 2002). Symptoms of E. Coli 0157: H7 Infection E.coli toxin can damage the lining of the intestine and cause other symptoms including Nausea , Several abdominal cramps , watery or very blood diarrhea , tiredness , vomiting (CDC, 2004) 26 Prevention of E.Coil 0157:H7 Infection. 1. Personal hygiene because the bacteria can be passed from one person to another if hygiene or hand washing habits is inadequate (CDC, 2004). 2. Adequate heat treatment for ground beef and milk. 3. Washing fresh fruits and vegetables thoroughly before eating raw or cooking them, (NIAID, 2002). 2.2.1.3- Clostridium Botulinum Infections Clostridium botulinum: is anaerobic, Gram positive spore forming rod that produces a potent neurotoxin. The spores are heat-resistant and can survive in foods that are incorrectly or minimally processed, (CDC, 2003). Seven types (A, B, C, D, E, F and G) of botulism are recognized based on the antigenic specificity of the toxin produced by each strain, types A, B, E, and F cause human botulism, types C and D causes most cases of botulism in animals. Animals most commonly affected are wild fowl and poultry, cattle, horses, and some species of fish (CDC, 2004). Clostridium spores are heat-resistant and can be destroyed only at temperatures above boiling. This is why canned food must be cooked to a high temperature under pressure as part of the canning process (NIAID, 2002). Toxins that cause botulism are completely inactivated by boiling. It can be destroyed at 80C˚ for 10 minutes or longer (CDC, 2003). 27 Botulism: is a rare serious paralytic illness caused by a nerve toxin that is produced by bacterium C. botulinum (CDC, 2004). Botulism occurs when the C. botulinum grows and produces a powerful paralytic toxin in foods. These toxins can produce illness even if the microbes that produced them are no longer there (CDC, 2003). According to CDC "in USA Health care providers report an average of 110 cases of food, infant and wound botulism to CDC each year, and about 10 -30 out breaks of foodborne botulism are reported every year (CDC, 2001). Out breaks involve two or more persons, and usually caused by eating contaminated home-canned foods (CDC, 2004). Infective Dose: a very small amount (few nanograms) of toxin can cause illness. The incidence of disease is low but the disease of considerable concern because of its high mortality rate if not treated immediately and properly .The onset of symptoms in food borne botulism is usually 18 to 36 hours after ingestion of the food containing toxins, although some cases have varied from 4 hours to 8 days (CDC, 2003). Associated Foods Almost any types of food that is not very acidic (pH above 4.6) can support growth and toxin production by C. Botulinum. Canned corn, peppers, green beans, soups, beets, mushroom, ripe olives, spinach, tuna fish, chicken, luncheon meats, sausage , smoked and salted fish (CDC, 2003).For more details, see Table ( 2 ). Symptoms 1. Double vision and dropping eyelids. 28 2. Slurred speech 3. Dry mouth and difficulty swallowing 4. Weak muscles. 2.2.1.4- Clostridium Perfringens Infections Clostridium perfringens is anaerobic, endospore former that produce a variety of toxins as well as gas during growth. It is the cause of gas gangrene. Some strains produce enterotoxins which cause food poisoning (Jawetz, Melnick, and Adelberg's, 2004). These microorganisms and their endospores have been isolated in many foods especially among red-meats, poultry, and sea food. Also from vegetable coated with soil or dust. People working in the kitchen may cross contaminate foods after using the toilet and not washing their hands. The incubation period is 8-22 hours and duration of illness is 12-24 hours symptoms; abdominal pains and diarrhea, the patient rarely vomits. Some endospores of this microorganism are killed in few minutes at 100C ْ◌ where as other require 1-4 hours at this temperature for complete destruction (CDC, 2004). Growth of Clostridium perfringens can be prevented by levels of curing salts and pH 6.2 or below (Gibson and Roberts, 1996). Prevention - Proper heat treatment of foods especially canned foods. - Proper refrigeration of food 29 2.2.1.5- Campylobacter Infections Campylobacter: is a bacterial pathogen that causes fever, diarrhea, and abdominal cramps. It is the most commonly identified bacterial cause of diarrhial illness in the world. These bacteria live in the intestines of healthy birds, and most raw poultry meat has been contaminated with juices dripping from raw chicken which is the most frequent source of this infection (CDC, 2003). Campylobacteriosis: is an infectious disease caused by bacteria of the genus campylobacter. Most human illness is caused by one species, called campylobacter jejuni, but 1% of human campylobacter cases are caused by other species (CDC, 2004). Other species like C. fetus and C. Coli can also cause compylobacteriosis usually occurs in a single, sporadic cases but it can also occur in outbreaks, when a number of people become ill at one time. Most cases of infections are associated with handling raw poultry or eating raw or under cooked poultry meat (CDC, 2004). According to CDC, "C. Jejuni is the leading cause of bacterial diarrheal illness in the united states, affecting an estimated 2.4 million people every year. The bacteria causes between 5 and 14 percent of all diarrheal illness world wide. C. Jejuni primarily affects children under 5 year's old and young adults (15-24 years old) (CDC, 2003). In a prospective case control study in Norway it is found that there are associated factors with an increased risk of Campylobacter infection these drinking undisinfectd water, eating at barbecues, eating poultry 30 bought raw, having occupational exposure to animals, and eating under cooked meat. The following factors were related to a decreased risk: eating mutton, raw fruits or berries, and swimming. Results indicate that infection is more likely to occur as a result of cross contamination from raw poultry products than because of poultry consumption" (Kapperud, and others, 2004). Symptoms of Campylobacteriosis 1. Diarrhea (often bloody). 2. Abdominal cramping and pain. 3. Nausea and vomiting 4. Fever and tiredness. Some infected people have no symptoms. Campylobacteriosis usually last for 2-5 days, but in some cases as long as 10 days (CDC, 2003). Prevention of Campylobacteriosis - Personal hygiene. - Proper cooking of poultry and poultry products. - Pasteurization of milk and milk products not less than 70C ْ◌. - Boiling of drinking water. - Proper cleaning and disinfection of preparation surfaces and utensils. 31 2.2.1.6- Staphylococcus Aureus Staphylococcus aureus: is a spherical bacterium (coccus) which on microscopic examination appears in pairs, short chain or bunched, grape- like clusters. These organisms are Gram-positive. Some strains are capable of producing a highly heat-stable protein toxin that causes illness in humans (CDC, 2003). Staphylococci exist in air, dust, sewage, water, milk and food or on food equipment and environmental surfaces. Humans and animals the primary reservoirs, Staphylococci are present in the nasal passages and throat and on the hair and skin of 50 percent or more of healthy individuals. (CDC, 2003). Staphylococcal Food Poisoning Staphyloenterotoxicosis is the name of the condition caused by enter toxins which some strains of S. aureus produce (CDC, 2003). According to FDA "An outbreak of staphylococcal foodborne illness was linked to canned mushrooms, growth and toxin production occurred prior to processing the mushrooms, without significant visual degradation, possibly because the mushrooms were held under ambient conditions in plastic bags with salt. Conditions within the bags rapidly become anaerobic and the toxin is heat stable" (FDA, 2001). Staphylococcus aureus can grow in some foods and produce a toxin that causes intense vomiting; this toxin cannot be inactivated by boiling (CDC, 2003). The onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute, depending on individual susceptibility to the 32 toxin, the amount of contaminated food eaten, the conc. of toxin in the food ingested, and the general health of the victim, (CDC, 2003). Associated Foods Foods that require considerable handling during preparation which are meats and meat products, poultry and egg products, salads, milk and milk products. Common Symptoms Nausea, Vomiting, abdominal cramping and prostration. In more sever cases, headache, muscle cramping and transient changes in blood pressure and pulse rate may occur (CDC, 2003). 2.2.1.7- Streptococcus SPP. The genus streptococcus is comprised of Gram-positive micro aerophilic cocci (round) which are not motile and occur in chains or pairs. The genus is defined by a combination of antigenic, hemolytic, and physiological characteristics into groups, A, B, C, D, F and G. group A and D can be transmitted to human via foods (CDC, 2003). Group A: sore and red throat, pain on swallowing, tonsillitis, high fever, headache, nausea, vomiting, malaise. Onset 1-3 days, the infectious dose is probably quite low (< 1000 organisms). Group D: Diarrhea, abdominal pain, nausea, vomiting, fever, chills, dizziness that occur in 2-36 hours after exposure the infectious dose is high (>107 organisms). 33 Associate Foods Group A food sources include milk, ice cream, eggs, steamed lobsters, potato, salads, egg salad, custard, rice, pudding. Entrance into the food is the result of poor hygiene, ill food handlers or the use of unpasteurized milk (CDC, 2003). Group D food source include sausage, evaporated milk, cheese, meat, raw milk. Entrance into the food chain is due to under processing or poor and unsanitary food preparation. 2.2.1.8- Shigellosis The genus shigella is composed of four species, shigella dysenteries, Shigella boydii, Shigella sonnei and Shigella flexneri. All species are pathogenic to humans at low dose of infection (FDA, 2001). Shigellosis is usually transmitted from person to person but many also occur by consumption of contaminated water and foods, including foods such as fruits or vegetables that have received little or no heat treatment. (FDA, 2001). Several large outbreaks of shigellosis have been attributed to the consumption of contaminated raw vegetables. According to FDA. "There are many out breaks associated with lettuce consumption. One of which was caused by S.sonnei that occurred simultaneously on two university campuses in Texas. On both campuses 111 students had eaten salads from self – serve salad bars, lettuce was the only produce item used in salad consumed by all students, who become ill". (Centers for Food Safety and Applied Nutrition & FDA, 2001). 34 In another out break of S.sonnei gastroenteritis was associated with eating shredded lettuce. All implicated restaurants received shredded lettuce from the same facility. An investigation suggested that a worker in the plant was the source of contamination and that the method of processing allowed contamination of the lettuce, (FDA, 2001). Two Midwestern United States outbreaks of S. Flexner infection have been linked to the consumption of fresh green onion. The onions were traced to shippers in California who obtained most of their green onions from a single farm in Mexico. It was concluded that contamination may have occurred in Mexico at harvest or during package .So according to FDA reports that "Shigella sonnei can survive on lettuce at 5C ْ◌ for 3 days without decreasing in number, and increased by more than 1000 fold at 22C ْ◌. Shigella can grow in shredded cabbage, chopped parsley stored at 24 C ْ◌ (FDA, 2001). See Table 2. 2.2.2 Viral Infections Viruses can be excreted in large numbers by infected individuals and have been isolated from sewage and untreated waste- water used for crop irrigation. Although viruses cannot grow in or on foods, but their presence on fresh produce, which may serve as vehicles for infections, is of concern (FDA, 2001). Hepatitis A Hepatitis A can be transmitted through food if handled in unsanitary conditions. 35 Hepatitis A virus (HAV) is a common form of acute viral hepatitis in many parts of the world. It is responsible for significant worldwide morbidity and occasional mortality. Outbreaks of hepatitis A occur periodically throughout the word, and fecaly contaminated food and water are the main vehicles (Bidawid, 2000). Hepatitis A is caused by hepatitis A virus (HAV).Transmission occurs by the fecal – oral route, either by direct contact with an HAV- in fected person or by ingestion of HAV- contaminated food or water (Fiore, 2004). According to Cruz and Oswaldo 2002 "there are three epidemiological patterns have been described world wide. In high endemic area with poor sanitary conditions, all children older than 10 years are immune to HAV. In these regions, outbreaks of the disease are uncommon and morbidity is low because in most children the infection is asymptomatic. In developed countries with better sanitary facilities, exposure to HAV during childhood is less common. In these areas, there are large numbers of susceptible adults creating the potential for out breaks. Finally developing countries demonstrate an intermediate endemicity with a growing number of adolescents and young a adults susceptible to HAV infection. This situation is due to the fact that improvements in sanitary conditions reduced the exposure to HAV in early childhood although the virus has not been eliminated from the environment". (Cruz, 2002). Symptoms of HAV: nausea, vomiting, abdominal pains, diarrhea, fever, hepatomegally, jaundice and darkening (Cruz, 2003). Table 2. Characteristics of some microbial pathogens that have been linked to out breaks of food illness. Microorg-Anism Incuba-Tion Period Symptoms Infectious Dose/(Number Of Cells) Source Clostridium Botulinum 12-36 hour Fatigues dizziness, dryness of mouth and throat, muscle paralysis difficulty of swallowing, double or blurred vision, drooping eyelids, and breathing difficulties. Death due to respiratory paralysis or Cardiac arrest Intoxication growth and toxin production in food. Soil lakes streams decaying vegetation Escherichia Coli 0157: H7 2-5day Bloody diarrhea, abdominal pain. Can lead to hemolytic uremic syndrome and kidney failure especially in children and the elderly. 10-1000 Animal feces, especially cattle, deer and human cross contamination from raw meat. Salmonella SPP 18 to 72 hour Abdominal pain, diarrhea chills, fever, nausea, vomiting. 10 to 100.000 Animal and human feces, cross contamination from raw meat, poultry, or eggs. Shigella SPP 1 to 3 day Abdominal pain, diarrhea, fever, vomiting. = 10 Human feces. Hepatitis A 25-30day Fever, malaise, anorexia nausea. 10 to 50 Human feces and urine Source: U.S. Food ad Drug Administration. (2001). 25 2.2.3 Yeast Yeast: the presence of yeast in foods creates spoilage and produce a slimy or cloudy appearance and by creating metabolic by products – especially through fermentation. They have widely different requirements for growth but generally require a high aw (is the amount of water available for deterioration reactions and is measured on a scale of 0 to l.0) than molds and yeast grow with or without oxygen and fermentation occurs under anaerobic conditions (Kuntz, 1996). 2.2.4 Molds Type of multicellular fungus, generates food spoilage with rot discoloration and off-flavor production. They may produce human toxins, as in the case of aflatoxins peanuts (Kuntz, 1996). Molds require free oxygen, a fairly high water old activity (aw), mesophilic temperature range (8-35C ْ◌) and as lightly acidic to neutral environment (Kuntz, 1996). 2.3 Factors Affecting the Growth of Microorganisms in Food Several factors encourage, prevent, or limit the growth of the microorganisms in food. 2.3.1- Foods: food is a chemically complex matrix. Most foods contain sufficient nutrients to support microbial growth, (FDA, 2003). The food substances are likely to be invaded by a variety of microorganisms. The type of food substances and the methods by which they are processed. Most of foods serve as good media for microbial growth. These microorganisms when given a chance to grow bring changes in natural 50 properties such as appearance, flavor, odor, taste, color, Etc, of the contaminated food thus causing spoilage (Lammerding and Paoli, 1999). Most bacteria prefer foods that have high content of protein and moisture like meats, poultry, seafood, dairy products, cooked rice, beans and potatoes. 1- Meat group a- Red Meats The interior portions of meat are usually free of microbial contaminations if healthy animals are properly slaughtered. The fresh cut meat gets immediately contaminated with microorganisms derived from gloves, hands, implements used to cut the meat, hides, hair, intestines of the animals and the environment of the slaughter house. Each new surface of meat, resulting from a new cut, adds more microorganisms to the exposed tissue, the most common microorganisms occurring on fresh meat include Clostridium, Escherichia Salmonella, and Streptococcus. (FDA, 2001). Consumption of red meats increased in Palestine during feasts, Ramadan, and special occupations (Hammad. 2003). b- Poultry The surface of freshly dressed eviscerated poultry has microbial flora, which is derived from the live birds or from the manipulations during killing, defeathring and evisceration. Species of Bacillus, Enterobacter, Escherichia, Salmonella and Staphylococcus constitute the major microbial flora on the skin of freshly dressed eviscerated poultry (Lammerding and Poali, 1999). 51 c- Eggs Clean eggs with uncracked shell normally don't contain microorganisms within. Poor sanitary and storage conditions under which it is held determine it's subsequent microbial content. Bacteria and Molds may enter the egg through cracks in the shell, (Lammerding and paoli, 1999). 2- Cereals Groups Cereals and cereal products contain microorganisms from insects, soil and other sources. Bacillus, lactobacillus, and micrococcus. Which are generally found on freshly harvested grains. Molds like Aspergillus, penicillum are also very common, (Lammerding and Poali, 1999). 3- Fruits and Vegetables Fresh fruits and vegetables can be contaminated if they are washed or irrigated with water that is contaminated with animal manure or human sewage (CDC, 2003). Generally the fruits are acid foods (pH below 4.5). While nearly all vegetables, fish, meat and milk products are non- acid foods (pH above 4.5) (FDA, 2001). During the production of fruits and vegetables. There are many points at which the produce can become contaminated with pathogens. The majority of them are related to contamination with pathogenic microorganisms through manure (for example through fertilization practices or run off contamination) water (for example irrigation water) and soil, (FDA, 2001). 52 2.3.2- Acidity (pH Value) There is a pH optimum for each microorganism at which growth is maximal. Moving a way from the pH optimum in either direction slows microbial growth (FDA and CFSAN, 2001). The pH affect the growth of microorganisms in food, most spoilage organisms prefer a pH in the slightly acidic to neutral ranges although proteolytic bacteria thrive in alkaline environments. By adjusting the PH, the spoilage can be a chivied, with a higher degree of protection from spoilage (Kuntz, 1996). 2.3.3 - Temperature Temperature values for microbial growth like pH values have a minimum and maximum range with an optimum temperature for maximal growth. The growth at extremes of temperature determines the classification of an organism (e.g. psychrotroph, thermotroph). The optimum growth temperature determines its classification as theromphile, mesophile, and psychrophile (FDA, 2001). Lowering temperatures results in a slowdown in the growth of organisms. Temperature below freezing stops the growth of most organisms. Freezing can be lethal to some microorganisms by formation of ice crystals (Kurtz, 1996). 2.3.4 - Time Bacteria reproduce by binary fission. Their multiplication therefore corresponds to a geometric progression: 20 21 22 23 2n. Generation time/time interval for one division of bacteria. As an example the generation time for E.Coli (doubling time) is 21 min at 40Cº (Schlegel, 1984). 53 However, when low acid food (food with a neutral or alkaline pH) is abused by placing it in the (5-60C) longer than two hours, pathogens multiply rapidly. Restricting the time that low acid foods stay in this temperature to two hours or less prevents growth of large numbers of pathogens, (Food Safety, 2004). 2.3.5- Oxygen In the relation of oxygen at least three groups of organisms can be distinguished. Obligate aerobes can obtain their energy only via aerobic respiration and are dependent on oxygen. Obligate anaerobes can grow only in the absence of oxygen; for these oxygen is toxic. Facultative anaerobes can grow either in the presence or absence of oxygen. Most foodborne disease causing microorganisms are facultative anaerobes (Shlegel, 1984). 2.3.6- Moisture All microorganisms must have an abundant supply of water to grow. Moisture content is the amount of water in food and expressed as a percentage. Water activity (aw) is the amount of water available for deterioration reactions and is measured on a scale of 0 to l.0. Bacteria, yeast and molds multiply rapidly at a high water activity (> 0.86). Meat products and soft cheeses have aw in this range (0.86 - 1.0) (Food Safety, 2004). Pathogenic bacteria have difficulty growing in foods such as jams and jellies, flours, etc. where aw is (< 0.85), because salt and sugar deprive microorganisms of water and inhibit their reproduction. These 54 products are shelf stable (they do not need refrigeration) (Food Safety, 2004). 2.4 Food Microbial Quality Indicators. 2.4.1 Total aerobic Count (TAC) The aerobic plate count indicating the level of microorganisms in a product and provides general estimate of live, aerobic, bacteria (excludes, obligate anaerobes), some times can be useful to indicate quality, shelf life and post heat processing contaminations (Maturin and peeler, 1998). . 2.4.2 Coliform Group (TC) and Fecal Coliform (FC) The coliform group contains all aerobic and facultative anaerobic, gram negative, non-spore forming rods able to ferment lactose with the production of acid and gas at 32 Cº or 35 Cº within 48 h, (Adams, 1995). The genera include: 1- Klebsiella: maybe found in feces and in the environment. 2- Escherichia: found always in human and other animal feces. 3- Enterobacter: found in feces and in the environment. 4- Serratia: found in environment. 2.4.3 Staph. Aureus 2.4.4 Salmonella 55 2.4.5 Yeast and Molds Coliforms can be Classified Into Fecal or Non-Fecal Origin The fecal coliform group is referred to as organisms that grow in the gastrointestinal tract of human and warm blooded animals and includes members of three genera: Escherichia, Klebsiella, and Enterobacter, (Adam, 1995). Fecal coliform bacteria, which belong to the entereobacteriaceae are present in large numbers in the feces and intestinal tracts of human and other warm-blooded animals and can enter water bodies from human abdominal waste. If large number of fecal coliform bacteria (over 200 colonies/100 milliliters (ml) of water sample) it is possible that pathogenic disease or illness causing organisms are also present in the water (WHO, 2000). 2.5 Factors Contributing to the Emergence of Food Borne Illnesses Outbreaks occur wherever pathogenic agents in sufficient number or quantity encounter a susceptible population without effective measures, (Hall, 1998). 1- Genetic Variability The large genetic variability of microorganisms is the principle reason why so often some microorganisms survive after any unfavorable environmental change. Some strains are hyper mutable, which reinforces the potential for survival and have very short generation times. (Hall, 1999). 56 2- Environment Environmental factors also contribute to emergence of foodborne illnesses; hot humid climates favor the growth of fungi and the production of mycotoxins (CDC, 1999). 3- Behavior (Travelers, Refugees, and Immigrants) Human actions and behavior directly affect food safety. People are vectors for disease, traveling from place to another more rapidly than ever before (CDC, 1999). According to WHO, it is estimated that about 90% of all cases of salmonella in Sweden are imported (WHO, 2002). 4- Urbanization Urbanization is a major factor in emergence. Crowding increase human contact and chances for transmission particularly in developing countries where the health services are far away from the villages and farms, so there will be gab in reporting the cases of outbreaks and investigations or disease surveillance will be very low, (Hall, 1999). 5- Denial A behavior that encourages outbreaks is denying the existence of an epidemic. This practice is more common in developing countries because they are concerned about the effect of outbreak publicity on tourist trade and exports (Hall, 1999). 6- Economics 57 War and economic collapse provide opportunities for disease outbreaks. The infrastructure that provides clean water, community medicine, disease surveillance, and food control all of these are easily affected by economic disruption (CDC, 1999). 7- Risk Factors These factors such as age, illness, behavior that promotes the incidence of diseases. 8- Interacting Factors The developing world, an interrelated and mutually reinforcing set of problems keeps foodborne disease at a high level as shown in figure 1. Figure 1. problems causing foodborne diseases in developing countries. Source: (Hall, 1999). Poverty Disease Malnutrition Contaminated food and water High Risk Groups that are Affected by Foodborne Illnesses. 1- Pregnant women. 2- The elderly 3- People with weakened immune systems 58 4- A bottle-fed infant is at higher risk for severe infections with salmonella or other bacteria that can grow in a bottle of warm formula if it is left at room temperature for many hours. 5- Persons with liver disease (CDC, 2003). 2.6 Important Definitions that are of Great Concern in this Field 1- Clean food or food-contact surfaces are washed and rinsed and are visually free of dust, dirt, food residues and other debris; (CDC, 1998). 2- Control measures any action or activity that can be used to prevent, reduce, or eliminate a microbiological hazard (CDC, 1998). 3- Sanitize to treat clean food by a process that is effective in destroying or substantially reducing the numbers of microorganisms of public health concern, as well as other undesirable microorganisms, without adversely affecting the quality of the food product or its safety for the consumer (CDC, 1998). 4- Food Code: contains standards for restaurants safety which is updated every 2 years, it includes temperature for cooking, cooling, refrigeration, reheating and holding food in food- service establishments (Collins, 1999). 5- cross contamination: is the transfer of bacteria from foods (usually raw) to other foods. The bacteria can transfer directly when one food comes in contact with another or indirectly for example from hands, equipments, tools, or working surfaces (NIH, 2003). 6- Hazard analysis: critical control points (HACCP). 59 Hazard analysis: identification of sensitive ingredients, critical processing points, and human factors that affect product safety, (Collins, 1999). Critical points/ processing determinants whose loss of control would result in an unacceptable food safety risk (Collins, 1999). Most contend that the HACCP system approach must be implemented at each stage of the farm- to – family continuum. Where are the critical control points and HACCP system development in the home, food service or retail establishments, or the car when food is carried from one location to another (Collins, 1999). In a study between 2001-2002 performed on 27 catering establishments in Ferrara (Italy) they took a total of 236 inspections after a HACCP system was introduced and educational programs for food staff was undertaken for approximately l0 years they found that the hygienic quality of services and foods has improved in comparison with previous surveys, showing that the staff educational programs and the application of HACCP principles have increased the level of awareness regarding food hygiene in those working in catering services (Kegnani and others, 2004). Table (4) summarizes Food born illness reports from restaurants in USA. Restaurants and Food Services Recent data indicates that 80% of reported foods borne illness outbreaks occur outside the home. Even though illnesses would be expected to be reported more, often they occur as a result of eating in restaurants (CDC, 1996). 60 2.7 Principles of Handling and Hygienic Control of Foods in Restaurants and Home. Food handling is an important factor in food safety. This includes the safety practices among those preparing and /or serving food as well as mode and duration of food storage. There are two main practices in food handling which increase the risk of foodborne diseases. First is preparation of food several hours before consumption and storage at temperatures that favor growth of pathogens and/or formation of bacterial toxins. Second is insufficient cooking or reheating of preserved food, (Walczak, 1998) Table 3. Food born illness reports from restaurants in USA, 1996. Date /Year No. of Cases Description Cause Junuary/1996 38 Salmonella Employees did not wash hands be fore handling food (contaminated food). Septemper/1995 11 Escherichia coli O154: H7 Raw food cross contaminated other "beef". August/1995 850 Salmonella Newport Raw chicken on cutting board with vegetables. January/1995 95 Hepatitis A Human fecal matter from handling hand washing. (Contaminated food). August./1994 56 Salmonella Holding temperature too low for 9hours (Hollandaise sauce). January/1993 7cases 1death Clostridium botulinium Un refrigerated storage of opened container (canned cheese sauce). Source: Center of science in the public interest, (1996). According to Atiya 2003 "the Palestinian, preventive medicine department MOH thought that the outbreaks occurred from restaurants 61 and houses meal, in Palestine can be considered a second major food poisoning. Recently, in 2002 outbreak occurred in hebron, where 51 cases of food poisoning were reported after consumption of soup and meat in a restaurant in Ramadan" (Atiya, 2003). In a case study conducted by Al-Khatib about microbial quality of food samples from restaurants and sweetshops in Ramallah and al-Bireh district. Utilizing food sample test results of Palestinian Ministry of Health, records to the years 1995, 1996, 2000, 2002, and the first 2 months of the year 2003, the results revealed that only 60.9%, 44.0%, 63.8%, 93.6%, 51.8%, 83.8%, and 50.4% of the food samples tested for total aerobic count, total Coliform, faecal Coliform, Staphylococcus b aureus, faecal Streptococci, moulds, and yeasts respectively are within the limits of the limits of the Palestinian and International standards. None of the tested samples for Salmonella or Clostridium perfringes were positive. The results of this study demonstrate a rise in the number of restaurants and sweet shops in the Ramallah/ al-Bireh urban area over time, ongoing and unacceptably high levels of contamination of tested food samples and the absence of clear guidelines and regulations for food handling in general. (Al-Khateeb, et.al. 2004) 2.8 General Rules to Reduce the Hazards of Foodborne Illnesses in Restaurants and Food Preparation Services. 1. The establishment must be provided with an approved water supply and sewage disposal system. 62 2. No person with symptoms or flu, gastrointestinal upset, throat, sinusitis, cough, infected cuts or sores or other contagious diseases shall work as a food handler or where food is prepared or served. 3. While on duty, all food service personnel must wear clean outer garments, maintain a high degree of personal cleanliness and conform to accepted hygienic practices, including proper hand washing. Suitable hair restraints must be used. Hand washing is recognized as the single most important measure to prevent cross contamination, because infected employees who work with food increase the risk of transmitting foodborne illnesses, (Worsfolds and Griffith, 2004). In an investigation about the transfer of HAV during contacts of hands Bidawid, farber and Sattar found that "Touching the lettuce with artificially contaminated finger pads for l0 sec at a pressure of 0.2 to 0.4 kg/cm2 resulted in transfer of 9.2% ± 0.09% of the infectious virus".(Bidawid ,Firebrand Sater , 2000) So the last experiments makes clearl the importance of hand washing and their role in cross contamination process and miss-handling can occur at any points In food chain, in processing, at supermarkets or restaurants, or in home (Collins, 1999). 4- Training There are many areas of training for the employees, they should train for: 1- The Importance of Good Hygiene 63 All personnel should understand the impact of poor personnel cleanliness and unsanitary practices on food safety. Good hygiene not only protects the workers from illness, but it reduces the potential for contamination of foods which if consumed by the public, could cause a large number of illnesses (FDA, 1998). 2- The Importance of Hand Washing Thorough hand washing before commencing work and after using the toilets is very important. Many of the diseases that are transmissible through food may be harbored in the employee's intestinal tract and shed in the feces. Contaminated hands can also transmit infectious diseases (FDA, 1998). 3- The Importance of Proper Hand Washing Techniques The workers should know how to wash their hands properly which include the following. - Hand washing with water. Warm water is more effective than cold water for washing hands - Use of soap. - Thorough scrubbing (including cleaning under fingernails and between fingers) rinsing and drying of the hands (FDA, 1998). 64 CHAPTER THREE 65 CHAPTER THREE METHODOLOGY Data about food samples microbiological quality from different sources and related possible food borne diseases was collected from the records of Nablus Health Directorate/Ministry of Health (MOH) for Nablus district for the years 1995-2003 with a population of 341,412 persons in 2004 (PCBS). The samples were collected for routine test of microbiological quality for public health issue by Environmental Health Inspectors of Nablus district. Also the maximum and minimum temperature and moisture degrees were collected for the years 1997-2003 from the Meteorology Department of Nablus district to observe the relation between the indicator microorganisms with temperature, moisture and food related disease. Numbers of food and sweets restaurants, food stores and food factories were collected from Nablus municipality and Nablus Chamber for Commerce and Industry .Interviews with the Environmental Health Inspectors in Nablus District and public health officials in Nablus municipality were done. 3.1. Samples Collection Samples were collected for analysis by Environmental Health inspectors in Nablus District and sent to central Public Health Lab in Ramalla for analysis. Microbiological food quality indicators which are (total aerobic count, Total Coliforms, fecal coliforms, Staphylococcus 66 aurous, streptococcus, Salmonella. Yeast and molds), as these are the only indicators tested by the MOH. Samples were collected for laboratory analysis at 0-4C˚ in clean and dry containers and transported on a weekly basis, and tested by the Central Public Health Laboratory, a body of the MOH in Ramallah. A sample unit consists of a minimum of 100 grams. The inspectors used sterile plastic cups of 200 gram, using the same spoon in which the workers of restaurants used, so the samples must be taken from the same dish in which people were served from. But for dry samples: randomly collected from many places of 100 grams, and kept in plastic containers. 3.2. Receiving Samples at the Laboratory As soon as samples arrive at the laboratory, time of collection and temperature are checked within 4 hours of receipt, samples are examined immediately or kept in the refrigerator for 24 hours of collection to be examined later. Samples are divided into three groups. 1- Frozen samples should be received frozen, and the temp should be below 0c ْ◌ preferably - 10 C˚ 2- Refrigerated samples should be received refrigerated with temp between 0-4 C˚ 3- Dry and canned samples should be received at ambient temp. This is between 15-25 C˚ 67 3.3. Samples Analysis A total of 1052 samples were coded and entered into the computer and analyzed using the statistical analytical system (SPSS). 3.3.1 Samples Categorization The samples were categorized and many cross tabulations were done like (food types, samples source, cooked or uncooked, with or without preservatives, years, months and seasons). 3.3.2 Sample Types The samples were categorized into eight groups. Fruit and vegetables, meat and chicken, cereal, diary product, salads, sweets, others and unknown and they were 18, 174, 27, 207, 32, 164, 332, and 55 samples respectively. Other includes (baking powder, vinegar, food additives, food colors, candies, jellies, tehenia, ect) or any food which contains more than two types of foods or mixture. 3.3.3 Samples Source Samples were collected by Environmental health inspectors from many food establishments, restaurants, supermarkets, houses, food factories, others, unknown, 61, 3, 1, 281, 4, and 646 samples respectively. Others includes (farms, schools, slaughterhouse) 3.3.4 Cooked or Uncooked Samples. 68 The samples also categorized according to the heat treatment, if the samples were cooked or not, the canned and pasteurized samples categorized as cooked. The numbers of cooked samples were 645, uncooked samples were 202 and others were 149 samples. Others includes (smoked foods, salted foods) 3.3.5 Samples With or Without Preservatives The samples also categorized into those that contain preservatives and those without preservatives. All canned foods categorized as with preservatives and other food items without preservatives and they were 14, 982 respectively. 3.3.6 Samples Distribution According to Years, Seasons and Months. The samples also categorized according to years, seasons, and months between the years 1995- 2003. 3.4 Acceptance of Samples Decisions regarding whether food samples were deemed acceptable or not acceptable for food preparation and/or human consumption were based on the Palestinian microbiological standards, WHO standards, and Gulf states standards, where each type of food is assigned a specific upper limit for the presence of microbes (Al-Khatib.2004) The bacteriological analytical manual online published by the U. S. Department of Health and Human Services (2001) was used as a reference for sample testing. 69 3.5 Methods of Detection of Microorganisms in Food Samples at Lab. 3.5.1 Detection of Total Coliforms in Diary and General Food Products. Procedure: add 50 g or 50 ml of test sample to200 1 peptone water, and then blend in a stomacher for one minute at medium speed. Make -1 to-3 dilutions in saline. Make duplicate plates on violet red bile lactose (Merck, USA) (VRBL) or EMB. Medium of each dilution spread plate technique, then incubate for 4 hours at 30c. Count colonies with diameter greater than 0.5 mm. And calculate the number of total Coliforms per gram or milliliter of sample. Subculture on BCP(MERCK, USA) and incubate for 4 hours at 30c.quality control: Both positive and negative organism control such as E. coli, Klebsiella, proteus and others, and negative media controls were included with each batch of samples. Confirmation: Kligler test, ONPG, Methyl red, Voges- prokauer, citrate, lactose fermentation and glucose fermentation. 3.5.2 Determination of Fecal Coliforms in Dairy and General Food Products. Procedure: Add 50g. or 50 ml of test sample to200 1 peptone water, then blend in a stomacher for one minute at medium speed. Make -1 to-3 dilutions in saline. Make duplicate plates on VRBL or EMB medium of each dilution by spread plate technique. Incubate for 18- 24 hours at 44 C˚, count red colonies with diameter greater than 0.5 m and calculate the number of fecal coliforms per gram or milliliter of sample. 70 Subculture on EMB Plates to Confirm E.Coli. Quality control: Both positive and negative organisms control should be included with each batch of samples. Confirmation tests: INDOL, ethyle red, voges-Proskaure, Citrate, Catalase, oxidase, glucose and lactose fermentation. 3.5.3 Detection of S. Aureus General Food Products. Procedure: add 50 g or 50 ml of test sample to200 peptone water, and then blend in a stomacher for one minute at medium speed. Make -1 to-4 dilutions in saline. Make duplicate plates of each dilution spread plate technique. Plates should be Baird – Parker agar incubated for 18-48 hours at 37 C˚. Count Black colonies with clear zone around, and calculate the number of bacteria in 1.0 g or 1.0 ml of sample. Incubate Brain Heart infusion broth and incubate for 24 hours at 37 C˚, to confirm with coagulase test. Quality control: Both positive and negative organism's controls and negative media controls were included with each batch of samples. Confirmation tests: Mannitol hydrolysis, Cagulase test. 3.5.3 Detection of Salmonella in General Food Products. Procedure: Add 25g. of test sample to 225ml, of peptone water or selenite cystine broth, Blend in stomacher for one minute at medium speed and, incubate for 16 hours at 37 C˚. 71 3.5.3.1 Isolation of Salmonella Add 0.1 ml of culture (peptone water) to 10.0 ml of Rappaport medium, (Meid) and incubate for 24 hours at 42 C˚. Selenite Medium (Merck, USA): Add 2.0 ml of culture (peptone water) to 20.0 ml of selenite cystine medium and incubate for 24 hours at 37 C˚. Quality control: Both positive and negative organisms' controls and negative media controls were included with each batch of samples. Confirmation tests: Biochemical identification and serological identification. Bizmuth Sulfite Plates: Inoculate 0.1 ml of rappaport medium or selenite or cystine medium onto bismuth sulfite plates for 24 hours at 37 C˚. Look for black colonies. 3.5.4 Detection of Yeast and Molds in General Food Products. Procedure: add 50 g or50 ml of test sample to200 peptone water, and then blend in a stomacher for one minute at medium speed. Make -1 to-4. Make duplicate plates on YGC medium (Merck, USA) dilutions in saline of each dilution by spread plate technique; per incubate at 22 – 25 C˚ for at least 5 days. Count colonies and 5 days. Examine colonies under microscope to differentiate between yeasts and molds, and Gram stain for yeasts. 72 CHAPTER FOUR 73 CHAPTER FOUR RESULTS 4.1. Distribution of Food Samples. Data of microbiological food examination recorded between 1995- 2003 at Environmental Health Department of Ministry of Health were analyzed and studied for microbial contamination in order to estimate the variations of bacterial quality of food by type, source, months, seasons, years. A total of 1052 samples where collected for routine test of microbiological quality for public health issue by Environmental Health Inspectors of Nablus district. The following tables show the distributions of these samples. 4.2. Microbial Quality and Food Types. Table (4) shows that out of 646 samples tested for Total Aerobic Count (TAC). Salads, meats, and diary products have the highest percentages of not accepted samples which were 18(62.1%) 8(14%), and 10(5.6%), respectively. The number and percentage of not accepted samples tested for Total Coliform (TC) for salads, fruit and vegetables, cereals ,diary products and meats, were 23(71.9%), 2 (11.1%), 3( 11.1%),21( 10.8%) ,and13( 7.5%), respectively. For Staphylococcus aureus (SA) the number and percentage of not accepted samples for meats, diary products and salads were 1(0.6%) 5(2.4%) and 1(3.1%) respectively. Meat and chickens, and diary products have the highest percentage of not accepted samples for Salmonella test which were 2.4% for Meat 74 and chickens and 0.5% for diary products. For yeast test the number and percentage of not accepted tested samples of meats and chickens, cereals, diary products and salads were 5(8.8%),1(25%), 11(5.6%) ,and 27(87.1%) respectively. The number and percentage of not accepted tested samples for Moulds for cereals, diary products salads and sweets were 1(25%), 4(2.0%), 4(12.9%) and 3(2.5%) respectively. 75 Table 4. Sample types and the acceptance of various microbiological tests S.aureusTC TAC Sample TotalNot Accept (%) Accepted (%) Total Not Accept (%) Accepted (%) Total Not Accept (%) Accepted (%) 12 0 (0.00) 12 (100) 18 2 (11.10) 16 (88.90) 2 0 (0.00) 2 (100) Fruit & Vegetable 172 1 (0.60) 171 (99.40) 174 13 (7.50) 161 (92.50) 57 8 (14.00) 49 (86.00) Meat &Chicken 23 0 (0.00) 23 (100) 27 3 (11.10) 24 (88.90) 3 0 (0.00) 3 (100) Cereal 207 5 (2.40) 202 (97.60 ) 194 21 (10.800) 173 (89.20) 178 10 (5.60) 168 (94.40) Diary Product 32 1 (3.10) 31 ( 96.90) 32 23 (71.90) 9 (28.10) 29 18 (62.10) 11 (37.90) Salad 127 0 (0.00) 127 (100) 164 7 (4.30) 157 (95.70) 120 2 (1.70) 118 (98.30) Sweets 245 0 (0.00) 245 ( 100) 332 30 (0.00) 302 (0.00) 212 23 (10.80) 189 (89.20) Other 53 0 (0.00) 53 ( 100) 65 11 (0.80) 54 (98.20) 45 1 (2.20) 44 (97.80) Unknown 871 7 (0.80) 864 (99.20) 996 100 (10.00) 896 (90.00) 646 62 (9.60) 584 (90.40) Total 147.462109.001Chi-Square 0.0000.000P- Value Note:- * accept means accepted . ** not accept means not accepted . *** TAC. Total Aerobic Count . ****T C. Total Coliform 76 Table 4. Continued Moulds Yeast Salmonella Total (%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted Sample 2 0 0.00 100 2 2 100 0 100 2 9 0.00 0 100 9 Fruit & Vegetable 57 0 0.00 100 57 57 5 8.80 52 91.20 168 2.40 4 97.60 164 Meat &Chicken 4 25.0 1 75.0 3 4 1 250 3 75.00 11 0.00 0 100 11 Cereal 197 2.00 4 98.0 193 197 11 5.60 186 94.40 207 0.50 1 99.50 206 Diary Product 31 12.90 4 87.1 27 31 27 87.10 4 12.90 32 0.00 0 100 32 Salad 122 2.50 3 97.5 119 122 4 3.30 118 96.70 123 0.00 0 100 123 Sweets 216 5.00 1 99.50 215 226 22 10.20 194 89.80 236 0.00 0 100 236 Other 45 0.00 0 100 45 45 1 2.20 44 97.80 45 0.00 0 100 45 Unknown 674 1.90 13 98.1 661 674 71 10.50 603 89.50 831 0.60 5 99.40 826 Total 35.685 209.390 Chi-Square 0.0000.000P- Value 77 4.3 Microbial Food Quality and Food Sources. Table (5) shows that out of 58 samples taken from restaurants and tested for TAC, the number and percentage of not accepted samples were 33(56.9%), while out of 257 samples taken from food factories and 323 samples of unknown source; the number and percentage of not accepted samples were only 6(2.3%) and 22(6.80%) respectively tested for the same test. Also table (5) shows that the number and percentage of not accepted samples taken from restaurants ,food factories and unknown source tested for TC were 40 (65.6%), 11(3.9%) and 48(7.4%) respectively. For Staphylococcus aureus the number and percentage of not accepted samples for restaurants, and unknown source were 1(1.6%) and 6(1.1%) respectively. Table (5) shows that the number and percentage of not accepted samples taken from food factories or of unknown source and tested for Salmonella were 1(0.4%) and 4(0.8%) respectively. For yeast test the number and percentage of not accepted samples from restaurants, food factories and unknown source were 44(75.9 %), 7(2.7%), and 20(5.7%) respectively. While the number and percentage of not accepted samples tested for Moulds taken from restaurants and unknown source were 3(5.2%), and 10(2.9%) respectively. 78 Table 5. Source of samples and acceptance of various microbiological tests. S.aureus TC TAC Source Total (%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted 61 1.60 1 98.40 60 61 65.60 40 34.40 21 58 56.90 33 43.10 25 Restaurants 3 0.00 0 100 3 3 0.00 0 100 3 3 0.00 0 100 3 Supermarket 1 0.00 0 100 1 1 0.00 0 100 1 1 0.00 0 100 1 Houses 267 0.00 0 100 267 281 3.90 11 96.10 270 257 2.30 6 97.70 251 Food Factory 4 0.00 0 100 4 4 250 1 75.00 3 4 25.00 1 75.00 3 Other 235 1.10 6 98.90 529 646 7.40 48 92.60 598 323 6.80 22 93.20 301 Unknown 971 0.80 7 99.20 864 996 10.00 100 90.00 896 646 9.60 62 90.40 584 Total 226.265 169.586 Chi-Square 0.0000.000 P- Value 79 Table 5. Continued Moulds Yeast Salmonella Source Total (%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted 58 5.20 3 94.80 55 58 75.90 44 24.10 14 61 0.00 0 100 61 Restaurants 3 0.00 0 100 3 3 0.00 0 100 3 3 0.00 0 100 3 Supermarket 1 0.00 0 100 1 1 0.00 0 100 1 1 0.00 0 100 1 Houses 260 0.00 0 100 260 260 2.70 7 97.30 253 264 0.40 1 99.60 263 Food Factory 4 0.00 0 100 4 4 0.00 0 100 4 4 0.00 0 100 4 Other 348 2.90 10 97.10 338 348 5.70 20 94.30 328 498 0.80 4 99.20 494 Unknown 674 1.90 13 98.10 661 885 71.00 28.2 89.50 603 831 0.60 5 99.40 826 Total 289.014 Chi-Square 0.000 P- Value 80 3.4. Microbial Quality and Cooked Foods. According to the results shown in table (6 ) the total number of cooked samples tested for TAC is 468 , of which 34(7.3%) were not accepted. For uncooked samples out of 75 samples tested for TAC 22(29.3%) were not accepted. For TC, out of 645 cooked tested samples, 56(8.7%) samples were not accepted, while out of 202 uncooked tested samples, 42(20.8%) were not accepted. For S. aureus, out of 566 cooked tested samples, 5(0.9%) samples were not accepted. However, out of 188 uncooked tested samples, 2(1.1%) were not accepted. For yeast, out of 492 cooked tested samples, 36(7.3%) samples were not accepted. While out of 78 cooked tested samples, 31(39.7%) were not accepted. For Moulds, out of 492 cooked tested sample, 7(1.4%) were not accepted and out of 78 uncooked tested samples, 6(7.7%) were not accepted. 81 Table 6. Numbers and percentages of acceptable, and unacceptable for cooked or uncooked samples S.aureusTC TAC Type Of Specimens Total(%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted 566 0.90 5 99.10 561 645 8.70 56 91.300 589 468 7.30 34 2.70 434 Cooked 188 1.10 2 98.90 186 202 20.80 42 79.20 160 75 29.30 22 70.70 53 Uncooked 117 0.00 0 100 117 149 1.30 2 98.70 147 103 5.80 6 94.20 97 Other 871 0.80 7 99.20 864 996 10.00 100 90.00 896 646 9.60 62 90.40 584 Total 39.652 38.293 Chi-Square 0.000 0.000 P- Value 82 Table 6. Continued Moulds Yeast SalmonellaType of Specimens Total (%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted 492 1.40 7 98.60 485 492 7.30 36 92.70 456 550 0.40 2 99.60 548 Cooked 78 7.70 6 92.30 72 78 39.70 31 60.30 47 174 1.70 3 98.30 171 Uncooked 104 0.00 0 100 104 104 3.80 4 96.20 100 107 0.00 0 100 107 Other 674 1.90 13 98.10 661 674 10.50 71 89.50 603 831 0.60 5 99.40 826 Total 16.409 80.952 Chi-Square 0.000 0.000 P- Value 83 4.5. Microbial Food Quality and Preservatives According to the results shown in table (7) the total number of samples with preservatives tested for TAC is 11, of which (0.0%) were not accepted. For samples without preservatives out of 635 samples tested for TAC 62(9.8%) were not accepted. For TC, out of 982 tested samples without preservatives 100(10.2%) samples were not accepted, while out of 14 tested samples with preservatives, 0(00.0%) were not accepted. For S. aureus, out of 857 tested samples without preservatives, 7(0.8%) samples were not accepted. However, out of 14 tested samples, 0(0.0%) were not accepted. For yeast, out of 663 tested samples without preservatives, 71(10.7%) samples were not accepted. While out of 11 tested samples with preservatives, 0(0.00%) were not accepted. For Moulds, out of 663 tested sample without preservatives, 13(2.0%) were not accepted and out of 11 tested samples with preservatives, 0(0.0%) were not accepted. 84 Table 7. Numbers and percentages of acceptable, and unacceptable samples with or without preservatives. S.aureusTC TAC Preservative Total(%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted 14 0.00 0 100 14 14 0.00 0 100 14 11 0.00 0 100 11 With Preservative 857 0.80 7 99.20 850 982 10.20 100 89.80 882 635 9.80 62 90.20 573 Without Preservative 871 0.80 7 99.20 864 996 10.00 100 90.00 896 646 9.60 62 90.40 584 Total Table 7. continued. Moulds Yeast Salmonella Preservative Total (%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted 11 0.00 0 100 11 11 0.00 0 100 11 14 0.00 0 100 14 With Preservative 663 2.00 13 98.00 650 663 10.70 71 89.30 592 817 0.60 5 99.40 812 Without Preservativ 674 1.90 13 98.10 661 674 10.50 71 89.50 603 831 0.60 5 99.40 826 Total 85 4.6. Numbers and Percentages of Acceptable and Unacceptable Samples during the Different Months of the Years 1995- 2003. Data presented in table (8) shows The number and percentage of not accepted samples for TAC test which were 22(28.6%) in July , 7(15.2%) in August, 6 (13.6%) in November , and 7(12.3%) in May . For TC test the number and percentage of not accepted samples were 19(22.1%) in May, 17(17.7%) in July, 15(14.7%) in April, and 12(18.5%) in August. For yeast test the number and percentage of not accepted samples were 18(30.5%) in May, 22(28.6) in July, 15(25.4%) in April, and 8(16.7%) in August. For Moulds The number and percentage of not accepted tested samples were 3(12.5%) in February 5(8.5%) in April, and 3(5.1%) in May. 86 Table 8. Numbers and percentages of acceptable and unacceptable samples during the twelve months of the years 1995- 2003. S.aureusTC TAC Month Total (%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted 50 0.00 0 100 50 72 2.80 2 97.20 70 33 3.00 1 97.00 32 January 37 0.00 0 100 37 54 1.90 1 98.10 53 21 0.00 0 100 21 February 49 0.00 0 100 49 48 2.10 1 97.90 47 31 3.20 1 96.80 30 March 87 2.30 2 97.70 85 102 14.70 15 85.30 87 53 9.40 5 90.60 48 April 82 3.70 3 96.30 79 86 22.10 19 77.90 67 57 12.30 7 87.70 50 May 70 0.00 0 100 70 76 10.50 8 89.50 68 53 9.40 5 90.60 48 June 88 0.00 0 100 88 96 17.70 17 82.30 79 77 28.60 22 71.40 55 July 60 1.70 1 98.30 59 65 18.50 12 81.50 53 46 15.20 7 84.80 39 August 106 0.00 0 100 106 111 6.30 7 93.70 104 97 6.20 6 93.80 91 September 69 1.40 1 98.60 68 87 5.70 5 94.30 82 52 3.80 2 96.20 50 October 87 S.aureusTC TAC Month Total (%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted 69 0.00 0 100 69 74 10.80 8 89.20 66 44 13.60 6 86.40 38 November 103 0.00 0 100 103 125 4.00 5 96.00 120 82 0.00 0 100 82 December 871 0.80 7 99.20 864 996 10.00 100 90.00 896 646 9.60 62 90.40 584 Total 47.828 52.237 Chi-Square 0.000 0.000 P- Value 88 Table 8. Continued Moulds Yeast SalmonellaMonth Total (%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted 34 2.90 1 97.10 33 34 2.90 1 97.10 33 39 0.00 0 100 39 January 24 12.5 3 87.50 21 24 12.50 3 87.50 21 35 0.00 0 100 35 February 31 0.00 0 100 31 31 3.20 1 96.80 30 46 2.20 1 97.80 45 March 59 8.50 5 91.50 54 59 25.40 15 74.60 44 75 1.30 1 98.70 74 April 59 5.10 3 94.90 56 59 30.50 18 69.50 41 81 0.00 0 100 81 May 57 0.00 0 100 57 57 0.00 0 100 57 66 0.00 0 100 66 June 77 0.00 0 100 77 77 28.60 22 71.40 55 88 0.00 0 100 88 July 48 0.00 0 100 48 48 16.70 8 83..30 40 58 0.00 0 100 58 August 99 0.00 0 100 99 99 1.00 1 99..00 98 106 0.00 0 100 106 September 54 0.00 0 100 54 54 1.90 1 98.10 53 68 2.90 2 97.10 66 October 48 2.10 1 97.90 47 48 2.10 1 97..90 47 68 0.00 0 100 68 November 89 Moulds Yeast SalmonellaMonth Total (%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted Total (%) Not Accept (%) Accepted 84 0.00 0 100 84 84 0.00 0 100 84 101 1.00 1 99.00 100 December 674 1.90 13 98.10 661 674 10.50 71 89.50 603 831 0.60 5 99.40 826 Total 39.360 105.375 Chi-Square 0.000 0.000 P- Value 90 4.7. Numbers and Percentages of Acceptable and Unacceptable Samples in Years 1995- 2003. Data presented in table (9) shows the number of tested samples for TAC test for the year 2002 were 97 and the number and percentage of not accepted samples for the same year were 12(12.4%) . In 2003 the number of samples were 549 with 50(9.1%) of not accepted samples for TAC test. For TC test, the number and percentage of not accepted samples tested for TC in 2003 ,2002, 1999, 1997 and 1995 were 61(10.9%), 11(10.8%) ,17(7.4%), 11(11.3%), and 0(0%) respectively. For Staphylococcus aureus The number and percentage of not accepted samples were 1(0.2%) 0(0.00%) ,5(3.6%),1(1.6), and 0(0%) respectively. For Salmonella, the number and percentage of not accepted samples were 1(0.2%) , 0(0.00%) , 1 (0.9%) , 3(7.0%) and 0(0%) respectively. For yeast, the number and percentage of not accepted samples were 49(8.7%), 13(12.6%), 8 (100%), and 1(100%) respectively. For moulds, the number and percentage of not accepted samples was 4(0.7%), 0(0.0%), 8 (100%), and 1(100%) respectively. Figure (5) shows the relationship between the years and the acceptance of Salmonella test. 91 Table 9. Numbers and percentages of acceptable and unacceptable samples in years 1995- 2003 S.aureusTC TAC Total(%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted Year 103 0.00 0 100 103 102 10.80 11 89.20 91 97 12.40 12 87.60 85 2002 562 0.20 1 99.80 561 562 10.90 61 89.10 501 549 9.10 50 90.90 499 2003 4 0.00 0 100 4 4 0.00 0 100 4 - - - - -= 1995 64 1.60 1 98.40 63 97 11.30 11 88.70 86 - - -- - - 1997 138 3.60 5 96.40 133 231 7.40 17 92.60 214 - - - - - 1999 871 0.80 7 99.20 864 996 10.00 100 90.00 896 646 9.60 62 90.40 584 Total 17.850 Chi-Square 0.001 p-value 92 Table 9. Continued Moulds Yeast Salmonella Total(%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted Year 103 0.00 0 100 103 103 12.60 13 87.40 90 103 0.00 0 100 103 2002 562 0.70 4 99.30 558 562 8.70 49 91.30 513 566 0.20 1 99.80 565 2003 - - - - - - - - - 4 0.00 0 100 4 1995 1 100 1 0.00 0 1 100 1 0.00 0 43 7.00 3 93.00 40 1997 8 100 8 0.00 0 8 100 8 0.00 0 115 0.90 1 99.10 114 1999 674 1.90 13 98.10 661 674 10.50 71 89.50 603 876 0.60 50 99.40 826 Total 464.042 78.878 31.716 Chi-Square 0.000 0.000 0.000 p- Value 93 4.8. Number and Percentage of Acceptable and Unacceptable Samples in the Four Seasons for the Years 1995-2003. Data presented in table (10) shows that the number of samples tested for TAC were 220, 194, 178, and 54 in Summer, Spring, Autumn, and Winter respectively. The number and percentage of not accepted samples were 35(15.9%) , 18(9.3%) , 8(4.5%) and 1(1.9%) respectively . also the table shows that 272 , 296, 286 , and 107 samples were taken in Summer , Spring , Autumn, and Winter respectively . The number and percentage of not accepted samples tested for TC were 36(13.2%) , 39(13.2%) , 18(6.3%) and 3(2.8%) respectively ,and 272 , 296, 286 , and 106 samples were taken in Summer , Spring , Autumn, and Winter respectively . For Salmonella the number of samples taken in Summer, Spring, Autumn, and Winter were 252, 259, 237 and 71 respectively. The number and percentage of samples tested for Salmonella were 1(0.4%) , 1(0.4%) ,3(1.3%) and 0(0.0%) for the seasons Summer , Spring , Autumn, and Winter . Data presented in table (10) shows that 224 , 204, 186, and 58 samples tested for yeast were taken in Summer , Spring , Autumn, and Winter respectively . The number and percentage of not accepted samples were 31(13.8%) , 32 (15.7%), 2 (1.1%)and 4 (6.9%). Also data presented in table (10) shows that 224, 204, 186, and 58 samples tested for moulds were taken in Summer, Spring, Autumn, and Winter respectively. The number and percentage of not accepted samples were 0(0.0%), 6 (2.9%), 1 (0.5%) and 4 (6.9%). 94 Table 10. Numbers and percentages of acceptable and unacceptable samples in the four seasons for the years 1995- 2003. S.aureusTC TAC Season Total (%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted 279 1.80 5 98.20 274 296 13.20 39 86.80 257 194 9.30 18 90.70 176 Spring 254 0.40 1 99.60 253 272 13.20 36 86.80 236 220 15.90 35 84.10 185 Summer 241 0.40 1 99.60 240 286 6.30 18 93.70 268 178 4.50 8 95.50 170 Autumn 83 0.00 0 100 83 106 2.80 3 96.30 103 54 1.90 1 98.10 53 Winter 857 0.80 7 99.20 850 960 10.1 96 89.90 864 646 9.60 62 90.40 584 Total 24.819.2 Chi-Square 0.000 0.000 p- value 95 Table 10. Continued Moulds Yeast Salmonella Season Total(%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted Total(%) Not Accept (%) Accepted 204 2.90 6 97.10 198 204 15.70 32 84.30 172 259 0.40 1 99.60 258 Spring 224 0.00 0 100 224 224 13.80 31 86.20 193 252 0.40 1 99.60 251 Summer 186 0.50 1 99.50 185 186 1.10 2 98.90 184 237 1.30 3 98.70 234 Autumn 58 6.90 4 93.10 54 58 6.90 4 93.10 54 71 0.00 0 100 71 Winter 672 1.60 11 98.40 661 672 10.30 69 89.70 603 818 0.60 4 99.40 814 Total 17.24 27.30 Chi-Square 0.001 0.000 p- value 96 4.9. Food Related Diseases in Nablus District. Data in table (11), (12) and (13) shows the monthly distributions of food related diseases in Nablus district for the years 2003, 2001 and 2000 For the year, 2003 table (11) shows that July, December and August, have the highest number of cases for hepatitis A which were 46, 40 and 39 respectively. While the cases of food poisoning were reported in June and May 25 and 13 respectively. While the high number of reported cases of food related diseases were concentrated in July, August and Jun 52, 50 and 49. For the year 2001, table (12) showed that April, September, and March, have the highest numbers of cases for food poisoning which were 10, 8 and 7 respectively. While the cases of Hepatitis A were reported in August, November and July 32, 30, and 29 respectively .While the high numbers of reported cases of food related diseases were concentrated in July, August and September 35, 37and 37. For the year 2000, table (13) show that June, August, and March, have the highest number of cases for food poisoning which were 22, 9 and 5 respectively. While the cases of Hepatitis A were reported in February, June, and August 55 ,35, and 34 respectively .While the high number of reported cases of food related diseases were concentrated in June, February, and August 58, 56 and 47. Table (14) shows the yearly distribution of food related diseases and the cases through the years 2003, 2001, and 2000 in which the total numbers of cases for food poisoning were 59, 43, and 44 respectively. While 355, 310 and 288 of Hepatitis A cases were reported. Also the total number of food related diseases was 420, 354 and 340 in the same years respectively. CHAPTER FIVE 100 CHAPTER FIVE DISCUSSION 5.1 Food Types and Acceptance of Various Microbiological Tests Data presented in table (4) shows that there is a significant relationship between the sample types and the acceptance of microbiological test TAC (P = 0.00, chi – square = 109.001). This study indicate that salads have the highest percentages of not accepted samples this is due to poor or inadequate hygienic-conditions during production, transportation, preparations and storage of salads and their constituents. The presence of large numbers of different kinds of bacteria indicates the possibility of food spoilage or food poisoning occurring. 5.1.1 Fruits and Vegetables In Nablus district, there is no control on the production of both fruits and vegetables, and there are many points at which products can become contaminated with human pathogens. The majority of these pathogens are related to manure through fertilization or irrigation water which is mainly sewage water like Wadi Altufah, Wadi al Zomar in western part of Nablus and Wadi – Al Badan. The contamination depends upon the product type, humidity, and temperature as well as the atmosphere and type of packaging. The source of contamination for raw fruits and vegetables with pathogenic organisms are namely: animals, insects, soil, water, dirty equipments and human handling. For example, according to CDC "fruits flies have been shown to transfer Escherichia Coli 0157:H7 to damage apples under laboratory condition" (CDC, 2001). 101 The transportation of fresh products from production places also plays an important role because of the obstacles imposed by the Israeli Army which places many barriers and checkpoints. This eventually leads to many physical damages to fresh products such as punctures or bruising on the epidermal barriers of fruits and vegetables, this will lead to contamination of fresh fruits and vegetables not to mention the effect of the environmental conditions like temperature and humidity which stimulate the growth and survival of pathogens especially during hot summer months. 5.1.2 Salads This study shows that salads have the highest percentages of not accepted samples nearly for all the microbiological tests. These findings are in agreement with the current situation of restaurants or food establishments. Poor hygienic practices like preparation of salads with naked hands without any regulations on preparation or refrigeration of salads, which are often left for many hours on show cupboard without any refrigeration or covering. During this period many pathogens can survive and multiply especially if there is no temperature control or bad storage conditions. Such conditions may occur during different incursions or curfew by the Israeli Army in the city. The result of the above-mentioned findings is in agreement with findings in a study conducted by FDA (2001) about the survival of viruses on vegetable salads " Hepatitis A, rotaviruses can survive in a variety of raw vegetables for period exceeding the normal shelf life of salad vegetables, survival appears to be dependent upon the pH , moisture content, and temperature ". These observations indicate that vegetable salads can serve as vehicles for the transmission of viral pathogens to humans. From the findings of this study, the weakness of the sampling system is obviously clear. Through the studied years only 32 102 samples of salad were tested, taking into account that the number of restaurants in Nablus city is about 228 restaurants in 2004. So, as a conclusion excessive handlings, improper preparation and storage, leads to high unacceptable percentages. 5.1.3 Meats and Diary Products This study indicates that meats and diary products have high percentages of unacceptable samples tested for TAC and Salmonella test, Meat and diary products are very rich media for microbial growth because of this they are immediately contaminated with microorganisms derived from globes, hands, implements used during processing or handlings after processing. In Nablus , the production of diary products like yoghurt and labanh is done at homes manually through many steps giving a chance for mishandling and contamination because the products are handled more often during preparation steps, and uncontrolled transportations. Recently Al-Safa plant began to produce diary products automatically with sterile automatic filling and packages. But still the traditional way is the mostly dominant. For meats, chickens, and eggs the control on production, transporting, marketing and storage are still limited. The municipality of Nablus controls the slaughtering of live animals in its slaughterhouse after the approval of the veterinary and health doctors, but in the rural areas of Nablus district there is no control over the slaughtering process. For chickens, slaughtering, defeathering and evisceration of the live birds are done in the marketing places. Furthermore eggs are held in poor sanitary and storage conditions for days in the same shops which are mainly used for marketing of chickens. The contamination by Salmonella can be 103 harmful if meats are eaten raw or partially cooked, so meat should be cooked to reach the temperature of 70C ْ◌ in deep. Meats, diary products and salads contain the highest percentages of unacceptable samples with total coliform. This gives a strong indication of fecal contamination due to inadequate processing and cross contamination due to poor hygienic practices of workers through food production chains or water contamination by sewage or contamination with human or animals feces in farms. Data presented in table( 4) showed that there is a significant relationship between the sample and the acceptance of yeast test ( p = 0.00, chi-square = 147.462) and the not accepted percentages of meats, cereals, diary products and salads which are 8.8%, 25%, 5.6% ,and 87.1% respectively. Presence of yeast in foods gives an indication of mishandling and inappropriate storage conditions. The growth of yeast gives the products a slimy or cloudy appearance by creating metabolic byproducts especially through fermentation. (Kuntz, 1996). Data presented in table (4) shows that there is a significant relationship between the sample and the acceptance of Moulds test ( p = 0.00, chi-square = 35.685). The not accepted percentage of cereals by moulds was high (25%). Cereals contamination by moulds is due to bad storage conditions with high moisture and temperatures inside the stores or shops, especially in the old city of Nablus. Grains, vegetables and fruits are susceptible to mould contamination prior to harvesting and during storage. 104 5.2 Acceptance of Sample Sources and Various Microbiological Tests. 5.2.1 Restaurants This study showed that restaurants in Nablus are suffering from high level of contamination, bad conditions, and poor hygienic practices for the following reasons:- 1- Economic status reduces the ability of the owners of restaurants to have a suitable clean water source and a reasonable bathroom with hygienic properties and proper sewage disposal. 2- Lack of experience because the