OCCURRENCE OF E. coli O157:H7 AND Salmonella sp. ON COOKED BURGER PATTIES SOLD AT POLYTECHNIC UNIVERSITY OF THE PHILIPPINES (NORTH AND EAST WING CANTEEN)

A Thesis Presented to The Faculty of the Department of Biology College of Science Polytechnic University of the Philippines Sta. Mesa, Manila

In Partial Fulfillment Of the Course Requirements for the Degree Bachelor of Science in Biology

MANGOMA, MILAGROSA C. BUENCONSEJO, ALVIN B.

2011

ACKNOWLEDGEMENT

This research study would not have been possible without support of many people who helped us. We would like to extend our gratitude to National Meat Inspection Services (NMIS) staff, the Director, Atty. Jane C. Bacayo, and Laboratory head, Ms. Rayne Bigay, Laboratory Staffs: Mrs. Remedios F. Micu, Ms.Evelyn Felicidad E. Embestro, Mrs. Meriam U.Nival, Mrs. Liza L. Nilo, Mrs. Maria Genoveva A. Borja, Sir Prudencio Denis, and the guards who lended us their laboratory facilities and without whose knowledge and assistance, this study would not have been successful. We owe our deepest appreciation due to Prof. Arcibel B. Bautista, our thesis adviser, who was abundantly helpful and offered invaluable assistance, support, guidance and patiently checked our papers and given her knowledge, time and valuable inputs and ideas in our study. Thank You very much Maam! To Prof. Joseph Aranas and Prof. Lincoln Bautista, made available their support in a number of ways and gives an advice on what to do in our study in terms of statistical analysis. To our Dear Panelist: Prof. Arnel O. Rendon, Prof. Ruthela P. Payawal, Prof. Carmelita P. Mapanao for their constructive inputs, suggestions and comments in our research. We indebted to many of B.S. Biology colleagues especially to Alejandro Jose Reboa, Adrea Ruiz, Michelle JC Doronilla, Charmaine dela Cruz, Larie Dianco,Dianna Rose Seva,Michelle Magcaling , and to Jomai Pascua to support and gave an inspiring uplifting advice throughout the years. To our beloved families especially our parents, for their understanding, endless love, support and financial in our study, through the duration of our study. And lastly to our almighty God, whod given us strength, courage, knowledge and love.

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Occurrence of E. coli O157:H7 and Salmonella sp. on Cooked Burger Patties sold at Polytechnic University of the Philippines (North and East wing canteen)

ABSTRACT

Hamburger patties, which play a significant role in modern diet, are core member of fast food family. It contained minced meat (either beef or beef and pork) sometimes with the addition of soya protein and generally bread crumbs. This study investigated the occurrence of Escherichia coli O157:H7 and Salmonella sp. on cooked burger patties sold at Polytechnic University of the Philippines (North and East Wing Canteen) and to identify the presence of the contamination of E. coli O157:H7 and Salmonella sp. Samples (n=72) were collected in three sampling period between 8 - 10 in the morning and undergo Microbiological Methods of Examination: determine the average pH, TPC (Total Plate Count) of cooked burger patties and Escherichia coli O157:H7 and Salmonella sp. Isolation and undergo a confirmatory/ Biochemical test respectively. Result showed that overall, the average pH of cooked burger patties from three sampling periods was 6.30 in which total bacterial count, 8.60 x100 was very low. Of the 72 cooked burger patties were negative on the occurrence of Escherichia coli O157:H7 and Salmonella sp. There is no need to test the significant difference between the distributions of Escherichia coli O157:H7 and Salmonella sp. in North and East Wing Canteen. The results shown that the time of sampling between 8-10 in the morning indicated that burger patties were refrigerated and freshly prepared cook, thus, cooking while burger patties are frozen avoid bacterial contamination. These suggest that burger patties that were being sold at the Polytechnic University of the Philippines (North and East Wing) canteen were clean and safe from Escherichia coli O157:H7 and Salmonella sp. Moreover recommended heat treatment of 70 o C for 15 second to 2 minutes or its equivalent is sufficient to give a reduction or destruction of pathogen and reduce the risks posed to consumers by the presence of burger patties. It recognized that apart from cooked burger patties, other food served must be undertake detection for the presence of pathogenic and also need to be addressed to reduce the public risk posed by this organism and should add more other indicator organisms that assess the quality and safety of foods and food ingredients

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TABLE OF CONTENTS Page No. Approval and Acceptance sheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List of Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Background Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Objectives of the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Significance of the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Scope and Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Review of Related Literatures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Food borne pathogens and Source of Contamination . . . . . . . . . . . . Hamburgers associated with food borne Pathogen . . . . . . . . . . . . . . E. coli O157:H7: Burger Bug and Environmental Pathogen . . . . . . . Virulence factors and Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . Salmonella sp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methodology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Microbiological methods of examination . . . . . . . . . . . . . . . . . . . . A.1. Weighing and cutting . . . . . . . . . . . . . . . . . . . . . . . . . . . A.2. Preparation of sample homogenate . . . . . . . . . . . . . . . . . A.3. pH determination of meat samples . . . . . . . . . . . . . . . . . A.4. Total Plate Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.E. coli O157:H7 Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.1. Confirmatory test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Salmonella sp. Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.1 Salmonella colony Morphology . . . . . . . . . . . . . . . . . . . . . C.1.1.Hektoen Enteric Agar (HEA) . . . . . . . . . . . . . . . C.1.2 .Xylose Lysine Desoxycholate (XLD) agar . . . . C.1.3.Brilliant-green Phenol-red Lactose sucrose agar (BPLS) . . . . . . . . . . . . . . C.2.Biochemical Test . . . . . . . . . . . . . . . . . . . . . . . . C.2.1.TSI and LIA Test . . . . . . . . . . . . . . . . C.2.2.Indole (I) Test . . . . . . . . . . . . . . . . . . . C.2.3.Methyl red (MR) Test . . . . . . . . . . . . . C.2.4. Voges-Proskauer (V) Test . . . . . . . . . C.2.5.Simmons Citrate agar (SC)Test . . . . . iv i ii iii iv vi vii 1 1 3 4 5 6 6 9 10 11 12 13 14 14 14 15 15 16 16 16 17 17 17 18 18 18 19 19 19 20

Page No . D. Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Schematic Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Occurrence of Bacteria on the Cooked Burger Patties . . . . . . pH of Burger Patties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Growth Occurrence on TPC of Cooked Burger Patties . . . . . . . . Summary, Conclusion, Recommendation . . . . . . . . . . . . . . . . . . . . . . Literatures cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 21 22 22 25 26 28 30 33

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List of Tables

Table No. 1 2 3

Title

Page No. 22 25 26

Occurrence of Bacteria on the Cooked Burger Patties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pH of Burger Patties . . . . . . . . . . . . . . . . . . . . . . . . Growth Occurrence on TPC of Cooked Burger . .

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List of Appendices Appendix No. A B Title Page No. 34 35 35 36 36 37 37 38 38 39 39 39 39 40 40 40 41 42 42 43

Letter of Request to Conduct at National Meat Inspection Service (NMIS) . . . . . . . . . . . . . . . . . . . . . . . . Preparation of Media: Buffered Peptone Water (BPW) . . . . . . . . . . . . . . . Tryptic Soy Broth + Novobiocin . . . . . . . . . . . . . . . Plate Count Agar (PCA) . . . . . . . . . . . . . . . . . . . . . Flourocult Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . Tetrathionate broth (TT broth) . . . . . . . . . . . . . . . . Rappaport-Vassiliadis (RVS broth) . . . . . . . . . . . . Brilliant-green Phenol-red Lactose Sucrose Agar (BPLS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xylose Lysine Deoxycholate(XLD) . . . . . . . . . . . . . Hektoen Enteric Agar (HEA) . . . . . . . . . . . . . . . . . Biochemical Test . . . . . . . . . . . . . . . . . . . . . . . . a. Tryptone Sugar Iron (TSI) . . . . . . . . . . . b. Lysine Iron Agar (LIA) . . . . . . . . . . . . . . c. Indole (I) . . . . . . . . . . . . . . . . . . . . . . . . d. Methyl Red (MR) . . . . . . . . . . . . . . . . . . e. Voges-Proskauer (VP) . . . . . . . . . . . . . f. Simmons citrate agar (SC) . . . . . . . . . . . Summary of Presumptive Results for E. coli O157:H7 . . . . Summary of Presumptive Results for Salmonella sp . . . . . . Raw Data for E. coli O157:H7 on Cooked Burger Patties. .

C C D E

Raw Data for Salmonella sp. on Cooked Burger Patties . . . 46

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CHAPTER 1 INTRODUCTION

Background Information Food preparation and safety are major issues in this modern world. These are especially important in the fast food sector. Hamburger patty, which plays a significant role in modern diet, is a core member of fast food family. Hamburger patties are often cooked in industrial hot grills, between two hot plates. The patties are cooked while frozen to avoid any bacterial contamination that may occur during the thawing process, due to the latent heat of melting of ice, the centre temperature of the patties may not rise to the necessary level for killing all pathogenic microorganisms. USDA-FSIS (1993) and FDA (1993) require that hamburger patties be cooked to 71 or 68.3 C and held for 15 seconds, respectively (Pan et al., 2000). Some food borne diseases are well recognized, but are considered emerging because they have recently become more common. Though there are various food borne pathogens that have been identified for food borne illness; Campylobacter, Salmonella, Listeria monocytogenes, and Escherichia coli O157:H7 have been generally found to be responsible for majority of food borneoutbreaks (Alocilja et al., 2003; Chemburu et al., 2005).

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Meats pose a direct food-poisoning hazard when consumed in a raw or undercooked form. However, a raw meat poses an indirect food-poisoning hazard through cross-contamination of cooked meats and other of other foods that are not cooked before consumption. It may also serve as a vehicle for the transfer of some antibiotic resistant microorganisms into foods where resistance could be passed both vertically and horizontally. This is due to contamination of food products and water by feces from animal and human origin. Fresh meat may carry Salmonella that caused diseases in the slaughtered animals or may be contaminated by handlers (Frazier et al., 1988). Salmonella sp have been found to be associated with illness diagnosed in many animals, including cattle with clinical signs of acute onset of fever and diarrhea. Among the enteric pathogens, Salmonella is considered to be the cause of the largest number of outbreaks, cases, and fatalities that result from food borne infections (Collins, 1995; Umholtz et al., 2002). An infectious dose of Salmonella is small, probably from 15 to 20 cells. E. coli O157:H7 is so-named because it expresses the 157th somatic (O) antigen identified and the 7th flagellar (H) antigen. The organism was first recognized as a human pathogen in 1982, when it was implicated in two outbreaks of hemorrhagic colitis, a distinctive clinical entity characterized by abdominal cramps, bloody stools, and a little or no fever (Paul S. Mead et al., 1998). It is important to note that extensive between animals cross

contamination with E. coli O157 takes place before dressing of slaughtered cattle

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i.e. during the lairage- to-stunning phase, through animalanimal and/or animal environmentanimal contact (Collis et al., 2004). Infectious dose of E. coli

O157:H7 ranging from 1 to 100 colony-forming units. These data show that it takes a very low number of microorganisms to cause illness in young children, the elderly and immune-compromised people (Snyder et al., 1998). To gave a safe assurance to the consumers who were buying the burger as a quick and easy to eat meal (immediate consumption) and at the same time fits for human consumption which is free from pathogenic bacteria.

Objectives of the study This study was undertaken to find out the presence Escherichia coli O157:H7 and Salmonella sp. on burger patties sold at PUP canteen (North and East Wing).Specifically this study attempts to: 1. detect the occurrence of Escherichia coli O157:H7 and Salmonella sp. cooked burger patties sold in the morning from 8 randomly selected stalls at Polytechnic University of the Philippines (North and East Wing Canteen);

2. identify the presence of the contamination of Escherichia coli O157:H7 and Salmonella sp.; and

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3.

determine the significant difference between the growth occurrence of Escherichia coli O157:H7 and Salmonella sp. in cooked burger patties from 8 randomly selected stalls at Polytechnic University of the Philippines (North and East Wing Canteen).

Significance of the Study The detection of selected pathogenic microorganism causing diseases in every burger patties sold at the Polytechnic University of the Philippines canteen gave a safe assurance of the consumer and to determine if the food handler were using quality control system .It is for this reason that the researchers were interested in taking more serious look into the health of the consumer specifically the students and the employees of Polytechnic University of the Philippines. The awareness of the students health thru this research may be the basis for the implementation of rules and regulation for the stall owner to improve their quality wholesome, and clean food.

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Scope and Limitations The study focuses only for the detection of selected pathogenic microorganisms such as Escherichia coli O157:H7 and Salmonella sp. of cooked burger patties sold specifically in the morning only at Polytechnic University of the Philippines Canteen (North and East Wing). 72 cooked burger patties samples were collected randomly in 8 stalls and three sampling period was done. The cooked burger patties were collected between 8:00 to 10:00 in the morning. Laboratory procedure and analysis will perform at the National Meat Inspection Service (NMIS) Central Laboratory for the detection of selected pathogenic microorganisms on cooked burger patties that sold at Polytechnic University of the Philippines canteen (North and East Wing). The confidentiality of the stall name and specific location treated strictly confidential by the researcher. The study limit only to the diseases associated exclusive only to Escherichia coli O157:H7 and Salmonella sp that can get from hamburger patties.

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CHAPTER 2 REVIEW OF THE RELATED LITERATURES

Food borne pathogens and Source of Contamination According to Tauxe et al., 2002 foods can be contaminated with microbes and the number of microbes present may be amplified at many points from growing or rearing on the farm to processing to final preparation. Foodborne pathogens which are always transmitted through food, while others are capable of being transmitted via several different routes in addition to food .Thus, to speak of foodborne disease is to speak of many pathogens and many diseases .Five foodborne pathogens such as E. coli O157:H7 ,Salmonella , Campylobacter , Listeria and Toxoplasma together cause estimated 3.5 million cases 33,00 hospitalization and 1600 deaths each year and yet many foodborne disease are preventable , though prevention will require a number of control efforts along the chain from production to consumption. Moreover, Li et al., 2004 stressed that meat and meat products have been implicated as significant sources of all pathogen, thus ground pork and beef, sausages, cattle carcass pork and lamb implicated as sources of contamination Strachan et al., 2006 reported that cattle and sheep as the major reservoir of E. coli O157:H7. On the other hand Chapman et al., 2001 considered cattle as the major reservoir of the pathogen which is carried in and excreted from their gastrointestinal tract without any symptoms of disease .Aslamet et al.,

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2003 considered feces and hides of cattle to be the main sources of E. coli O157 contamination of carcasses during slaughter. Collis et al., 2004 evaluated that it is important to note that extensive between animals cross-contamination with E. coli O157 takes place before dressing of slaughtered cattle during the lairage- tostunning phase, through animalanimal and/or animalenvironmentanimal contact. Chapman et al., 1995 and Hancock et al., 1998 stressed that contamination of carcass meat with E. coli O157 can occur during dressing; primarily during the skinning but also during the evisceration phase. In addition to cattle, E. coli O157: H7 has been isolated from deer, goats, dogs, birds, and flies. Thus, Hernandez et al., 1998 reported that in Spain, from 1993 to 1998, 49% of foodborne outbreaks occurred in the household environment, Salmonella being the main pathogen (60%) followed by Staphylococcus sp. These data showed that consumers often failed to store, handle and prepare food in a hygienic and safe manner. Moreover, studies such as those of Gorman et al., 2002 have demonstrated the ability of pathogens such as Staphylococcus aureus, Escherichia coli, Salmonella and Campylobacter to be transferred from contaminated food to hand and food contact surfaces in the kitchen. Beumer and Kusumaningrum (2003) and Bloomfield and Scott (1997) stated that cross contamination in the kitchen environment can be a significant

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factor in the household outbreaks. Reij et al., 2004 reviewed scientific literature on recontamination routes and sources (e.g. raw materials, food contact surfaces, food handlers, etc.) and demonstrated their relevance to foodborne disease outbreaks. They also suggested that such knowledge on

recontamination should be incorporated into exposure assessments of Quantitative Microbiological Risk Assessments (QMRA) to help in determining mitigation strategies to reduce foodborne disease. Thus, the importance of performing QMRA (Codex Alimentarius Commission, 1999) has led to researchers to try and quantify cross contamination events. Recent outbreaks prompted by Wachtel (2003) caused by E. coli O157:H7 associated with cross contamination between raw meat and raw vegetables to quantify the transfer of E. coli O157:H7 from raw contaminated meat to hands and cutting surfaces, and then to iceberg lettuce by contaminated hands and cutting boards. They studied the effect of successive contacts (cutting lettuce leaves with a contaminated knife) on whether or not and how much was transferred. The result revealed the random nature of cross contamination events when they used low inoculum.

Hamburgers Associated with food Borne Pathogen According to Tamminga et al., 1982 that the hamburgers contained minced meat (either beef or beef and pork) sometimes with the addition of horse meat or soya

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protein and generally bread crumbs. Hamburger patty, which plays a significant role in modern diet, is a core member of fast food family. In study of Cagney et al., 2004 2.80 percent were positive for E. coli serotypes. The study was found that E. coli O 157:H7 was found in almost all products (ground beef, burger patties) including fresh unpacked minced and burgers, fresh packed minced and burgers and also frozen burgers. Kassenborg et al., 2004 identified that hamburger patties are often cooked in industrial hot grills, between two heated plates. The patties are cooked while frozen to avoid any bacterial contamination that may occur during the thawing process. Due to the high latent heat of melting of ice, the centre temperature of the patties may not rise to the necessary level for killing all pathogenic microorganisms. USDA-FSIS (1993) and FDA (1993) require that hamburger patties be cooked to 71 C. Many outbreaks of E. coli O157:H7 has been linked to beef burgers and analyses of sporadic cases of E. coli O157:H7 infections. Strachan et al., 2006 claimed that case control studies indicate strong risk associates with environmental exposure in Scotland; the United Kingdom (UK) as a whole and the United States of America (USA), but burgers appears to be more risk in the USA.

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Escherichia coli O157:H7: Burger Bug and Environmental Pathogen According to Mead, 1998 that E. coli O157:H7 was first identified as human pathogen in 1982. It is one of the several shiga toxin producing serotypes known to cause human illness. E. coli O157:H7 is found regularly as mention

previously in the feces of healthy cattle, and is transmitted to human through contaminated food, water and direct contact with infected people or animals. Strachan et al., 2006 considered that E. coli O157:H7 associated with

foodborne from eating contaminated burgers and has been termed the burger bug in the popular press. Moreover, analysis of E. coli O157:H7 outbreaks in Scotland from 1992 to 2003 associated either meat or dairy products or with environmental transmission shows that approximately 40 percent of the outbreak were foodborne, 55 percent were environmental and 6 percent involved both transmission routes . Thus, Karmali et al., 1983 reported an association between infections with Escherichia coli 0157:H7 that produce shiga toxin and post diarrhea Haemolytic Uremic Syndrome (HUS), a clinical condition defined by acute renal injury, thrombocytopenia, and microangiopathic haemolytic anemia.

Virulence Factors and Pathogenesis Mead, 1998 specified that among the most important virulence characteristics of Escherichia coli 0157:H7 is ability to produce one or more shiga toxin. Strachan et al., 2006 and Signorini et al., 2009 reported that shiga toxin is

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also called as verocytotoxins (Verocytotoxigenic Escherichia coli 0157:H7 or VTEC) and Shiga toxin producing Escherichia coli 0157:H7 (STEC) .Shiga toxin 1 is related in Shigella dysenteriae type 1 and shiga toxin 2 is produced most of Escherichia coli 0157:H7 .Shiga toxin 2 is more divergent molecule with only 56 percent aminoacid homology. Both toxins are composed of five b subunits and single sub units and are encoded on a temperate bacteriophage inserted into Escherichia coli 0157:H7 chromosome. Shiga toxins have both local and systemic effects on the intestine and are probably critical to the development of bloody diarrhea. Thus, postdiarrhea Haemolytic Uremic Syndrome HUS is primary diseases of microvasculature though to develop when shiga toxins produced in the intestine enter the bloood and bind to GB3- rich endothelial cells in the kidney .Damage of endothelial cella in the kidney, indicated by shiga toxins, may trigger platelet and fibrin deposition leading to injury of passing erythrocytes (haemolysis) and occlusion of renal microvasculature (renal failure). Organs including brain may be affected, resulting in a wide range of complications.Cieslak et al., 1997 stressed that at least 58 persons became ill with bloody diarrhea in Las Vegas, Nevada, in December 1992 and January 1993; 3 of these individuals developed hemolytic uremic syndrome. As in the concurrent Washington State outbreak,

epidemiologic evidence strongly implicated consumption of regular (0.1 lb) hamburgers at fast food chain. Cooking procedures at the time of the outbreak were frequently inadequate to kill E. coli 0157:H7.14 the hamburgers were obviously cooked quite unevenly, as demonstrated by the variability of

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temperatures recorded within a single hamburger. It indicates the difficulty in cooking hamburgers adequately to kill pathogens.

Salmonella sp. On the other hand Nyeleti et al., 2000 evaluated the incidence of salmonellosis as a result of consumption of meat obtained from unhealthy animals. Humans become infected with Salmonella primarily through fecal contamination of food products or water. Another source of human infection, primarily affecting farm families, employees, and visitors are with contact with ill animals (Wells et al., 2001). Due to the contamination of food products and water by feces from animal and human origin fresh meat may carry Salmonella that caused diseases in the slaughtered animals or may be contaminated by handlers (Frazier & Westhoff, 1988). Mremu et al., 2006 stated that Salmonella typhi and Salmonella anatum are isolated in burger patties. Typical symptoms of illness associated with salmonellasis include nausea, vomiting, and diarrhea. Doust et al., 2001 added that complications associated with infection include septicemia realtice arthritis.

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CHAPTER 3 METHODOLOGY The laboratory procedure and analysis performed at National Meat Inspection Service (NMIS) Central laboratory for the detection of occurrence of E. coli O157:H7 and Salmonella sp. on cooked burger patties that sold at the Polytechnic University of the Philippines Canteen (North and East Wing). Collection of samples taken in the morning from eight (8) stalls of Polytechnic University of the Philippines Canteen (North and East wing).Seventytwo (72) cooked burger samples were collected in three (3) sampling period. Collection of samples was done in one day for each sampling period and randomly purchased from eight (8) stalls. A total of twenty four (24) samples were taken from eight(8) stall from each sampling period. During visit to the sampling site, 3 cooked burger samples taken from each stall between 8 to 10 in the morning, placed in ziplock and into the ice box, then samples transported immediately to National Meat Inspection Service (NMIS) to subject laboratory analysis. The Microbiological Methods of Examination was adapted with existing manual that was used in the National Meat Inspection Service (NMIS) that were followed to accomplish this research.

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A. Microbiological Methods of Examination A.1. Weighing and cutting A total of 21 g of meat samples were needed for the study .Aseptically, 1g and 10 g of meat sample were needed for Escherichia coli 0157:H7 and Salmonella sp. respectively, and another 1 g of meat for total plate count, then minced in a foil using sterile scissors with the help of forceps .Another 10 g of meat samples were weigh and cut for pH determination A.2. Preparation of sample homogenate In a sample bottle 90 ml of Buffered Peptone Water (BPW) were needed to 10 g of meat samples for Salmonella sp. For E. coli O157:H7, 1g of meat sample added in a sample bottle with 10 ml Tryptic Soy Broth + Novobiocin ( TSB + Novobocin).The dilution was mixed and shaked in a sample bottle 25 times in a 30 cm arc approximately 7 sec as a results of dilution 10-1

.For the

Total Plate Count, a 1:10 dilution of the sample was prepared aseptically by adding the analytical unit into 9 ml of Buffered Peptone Water (BPW) diluents and transferred 1 ml of the previous dilutions into 9 ml of 0.01 percent Buffered Peptone Water (BPW) .The same procedure was done to the rest of the tubes .Each dilution was shaked again immediately prior to making transferred to

ensure uniform distribution of the microorganism present . Separate pipette were used for making each transfer.

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A.3. pH determination of meat samples 10 g of meat sample homogenized and were added in 50 ml beaker with 40 ml distilled water. The pH of the samples were measure at room temperature (approximately 25 C) A.4. Total Plate Count a. Pour method Agitated each dilution to resuspend materials and pipetted 1 ml of each prepared dilution into each three appropriate 3 marked petri plates. The Plate Count Agar (37o C) was added in the plates. The dilutions were mixed thoroughly and uniformly by alternate rotation and back-and-forth motion of plates on flat level surface. The agars were wait to solidify and inverted the petri dishes, and incubated promptly for 48 2 h at 35C. Then, the colonies were counted promptly after the incubation period. To compute the number of colony, express as colony forming unit (cfu/ g), the formula was used (Maturin et al., 1998): N = C / [(1 * n1) + (0.1 * n2) + (0.01*n3] * (d) Where N = Number of colonies per ml or g of product C = Sum of all colonies on all plates counted n1 = Number of plates in first dilution counted

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n2 = Number of plates in second dilution counted n3= Number of plates in third dilution counted d = Dilution from which the first counts were obtained

B.E. coli O157:H7 Isolation Previous dilutions were incubated at 37o 2 for 15 to 24 hrs. The dilution was streak after the incubation period to fluorocult agar and incubated again at 37 o 2 for 15 to 24 hrs. Colorless colony on the surface of the media was determined as possible for E. coli O157:H7 and subjected to ultraviolet lights and observed for the fluorescence color of the colony. The plates were not fluorescence was subjected to confirmatory test. B.1.Confirmatory test A drop of E. coli O157:H7 reagent was placed on the glass slide. Using a sterile inoculation loop, a large colony was picked of presumptive positive E. coli O157:H7 and mixed with the reagent. Agglutination or precipitated within 2- 3 minutes occurred for positive results of E. coli O157:H7. C. Salmonella sp. Isolation Dilution was incubated at 24 2 hrs at 37o

C. After the 24 hr incubation

the lid was tighten and gently shaked the individual samples .1 ml mixture was

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tranferred to Tetrathionate broth (TT broth) and 0.1 mixtures to 10 ml RappaportVassiliadis medium. Selective media was incubated as follows: -RVS medium at 24 2 hrs at 42 o C -TT medium at 24 2 hrs at 35 o C A vortex was used to mixed the incubated Tetrathionate broth (TT) and Rappaport-Vassiliadis (RVS and streaked 3mm loopful on Brilliant-green Phenolred Lactose Sucrose (BPLS), Xylose Lysine Deoxycholate(XLD) , and Hektoen enteric Agar ( HEA ). C.1. Salmonella Colony Color C.1.1. Hektoen Enteric Agar (HEA). Colonies appeared to be blue-green to blue colonies with or without black centers. Many cultures of Salmonella may produce colonies with large, glossy black centers or may appear as almost completely black colonies. C.1.2. Xylose Lysine Deoxycholate (XLD) Agar. Color colonies appeared to be pink colonies with or without black centers. Many cultures of Salmonella may produce colonies with large, glossy black centers or may appear as almost completely black colonies.

18

C.1.3 Brilliant-green Phenol-red Lactose Sucrose (BPLS). Pink colonies with or without black centers for Salmonella were be observed on the surface of the media. The presumptive positive Salmonella shown on the surface of the medium were preceded to biochemical test. C.2. Biochemical Test C.2.1. Tryptone Sugar Iron (TSI) and Lysine Iron Agar ( LIA) Test

The center of the colony

was picked with sterile inoculating needle and

inoculate Tryptone Sugar Iron (TSI) slant by streaking slant and stabbing butt. Without flaming, Lysine Iron Agar (LIA) was inoculated the slant by stabbing butt twice and streaked the slant. Tryptone Sugar Iron (TSI) and Lysine Iron Agar (LIA) slants was incubated at 35C for 24 2 h. Salmonella in culture typically produces alkaline (red) slant and acid (yellow) butt, with or without production of H2S (blackening of agar) in Tryptone Sugar Iron (TSI). In Lysine Iron Agar (LIA), Salmonella typically produces alkaline (purple) reaction in butt of tube.. Most Salmonella cultures produce H2S in Lysine Iron Agar (LIA). C.2.2. Indole Test From the inoculated unclassified TSI AND LIA, the loop was dipped to 5 ml of 24 h tryptophane broth and incubated at 35C for 24 2 h. 0.2-0.3

ml Kovacs' reagent was added if and only if the TSI AND LIA was gave a

19

respective

color

of Salmonella on the slant and butt on the media .

Most Salmonella cultures give negative test (lack of deep red color at surface of broth). C.2.3. Methyl red Test (MR) From the inoculated Indole test the loop was dipped to 5 ml MR-VP broth and incubated at 35C for 24 2 h. 5-6 drops of methyl red indicator was added if and only if Indole test gave a respective color for the Salmonella. Most Salmonella cultures give positive test, indicated by diffuse red color in medium. A distinct yellow color was negative test. C.2.4. Voges-Proskauer (VP) Test From the inoculated Methyl red test , the inoculating needle was dipped

to 1 ml 48 h culture .0.6 ml -naphthol was added if and only if the Methyl red test was gave a respective color for the Salmonella and 0.2 ml 40% KOH

solution was added and shaked the test tube . To intensify and speed reaction, few crystals of creatine were added to the solution. Result was read after 4 minutes which developed of pink-to-ruby red color throughout medium is positive test. Most cultures of Salmonella are VP-negative, indicated by absence of development of pink-to-red color throughout broth.

20

C.2.5 Simmons citrate agar (SC) Test From the VP Test the inoculating needle was streak and incubated at 24 2 h at 35C.Positive results shows presence of growth, usually accompanied by color change from green to blue. Most cultures of Salmonella are citrate-positive. Negative results indicate no growth or very little growth and no color change.

D .Statistical Analysis To compare the results, two independent sample t-tests (equal sample sizes, equal variance) are to be use in SPSS version 17.0 software to differentiate the distribution of Escherichia coli O157:H7 and Salmonella sp. in North and East Wing Canteen and p > 0.05 significance was used.

21

72 samples are to be collected randomly from 8 stall inside the PUP canteen (North and East Wing) canteen

The samples are to be transported at National Meat Inspection Service (NMIS) for subject laboratory examination Microbiological Examination

pH determination and Total plate count on meat samples E. coli O157:H7 Isolation Salmonella sp. Isolation

Confirmatory Test

Biochemical Test

Statistical Analysis

Figure No. 1 Schematic diagram of methodology.

22

CHAPTER 4 RESULTS AND DISCUSSIONS

Occurrence of Bacteria on the Cooked Burger Patties Of the 72 cooked burger patties, there were no occurrences (absent) of Escherichia coli O157:H7 and Salmonella sp. sampled in the morning from the 8 randomly stall collected at the Polytechnic University of the Philippines Canteen (North and East Wing canteen). The absence of E. coli O157:H7 and Salmonella sp. indicates that time of sampling between 8-10 in the morning that burger patties was stored and refrigerated and freshly prepared cook, thus, cooking while the burger patties were frozen avoid bacterial contamination. Considering the number of patties sold during the sampling period on month of December was small. These suggest that in this study burger patty is clean and met the appropriate temperature and time of cooking and that is safe from Escherichia coli O157:H7 and Salmonella sp. Risk

Table 1.Presence

of E. coli O157:H7 and Salmonella sp. in cooked burger patties. Bacterial Analysis Occurrence E. coli O157:H7 absent Salmonella sp. absent

23

Moreover recommended heat treatment of 70

o

C for 15 second to 2

minutes or its equivalent is sufficient to give a reduction or destruction of pathogen and reduce the risks posed to consumers by the presence of burger patties (Byne et al., 2004). The result of Escherichia coli O157:H7 in cooked burger patties was different than noted in previous studies like 2.80 % for 43 out of 1533 of samples (Cagney et al., 2004), and 3.30% for 43out of 1303 (Crowley et al., 2005). For the comparison on the result of Salmonella sp. 7.4% or 4 out of 58 of samples ( Mrema et al., 2006 ) was being observed . However, taking sample size into account, direct comparison of results is difficult due to differences in the study methodologies such as sampling size and type of the sample to be analyzed. Previous research used raw sample burger patties while in this current studies, cooked burger sample were analyzed and focused to determine the occurrence of Escherichia coli O157:H7 and Salmonella sp. in such a way direct/immediate consumption will be observed. In the study of Duitshaever, Bullock and Arnott (1977) did not detect Salmonella, and in which total bacterial counts were usually lower than 104 per g contrarily to this study on bacterial counts which is much lower , but Mueller

24

(1975) reported salmonellas in 3 of the 13 pre-cooked hamburger samples, and total counts between 104 and 105 per g. Microbiological method tested on E. coli O157:H7,Tryptic Soy Broth + Novobiocin ( TSB + Novobocin) was used as pre- enrichment selective broth and isolated in fluorocult agar as selective media .After the incubation period it was resulted from the surface of the agar a yellow colony from some plates while the other plates gave a black colony on the surface of the media indicates a negative result of E. coli O157:H7. Presumptive positive plates of E. coli O157:H7 results showed a colorless colony and then subjecting to ultra violet lights to determine the fluorescence of the colony. The colony on the plates was fluorescence indicates a negative colony for E. coli O157:H7 .Plates with presumptive E. coli O157:H7 was not fluorescence, and proceeded to agglutination test where a drop of E. coli O157:H7 was added .Precipitation or agglutination was not observed within to 3 minutes indicates that the colony was not E. coli O157:H7. In Salmonella sp. Buffered Peptone Water (BPW) was used for the preliminary enrichment of bacteria, followed by Tetrathionate broth (TT broth) and Rappaport-Vassiliadis medium as selective broth only for Salmonella sp. After the specified incubation period, Hektoen Enteric Agar (HEA), Xylose Lysine Deoxycholate (XLD) Agar and Brilliant-green Phenol-red Lactose Sucrose (BPLS) was used as a three differential media in order to isolate Salmonella sp. on cooked burger patties. Xylose Lysine Deoxycholate (XLD) Agar and Brilliant-

25

green Phenol-red Lactose Sucrose (BPLS) was observed for a pink colony with or without black center as presumptive positive results for Salmonella sp., while Hektoen Enteric Agar (HEA) was observed for blue green colony on the surface of the media. Presumptive positive results on each differential media were preceded to Biochemical Test. Biochemical examination include Tryptone Sugar Iron (TSI) where slant was inoculated gave a red color and yellow butt while Lysine Iron Agar ( LIA) Test , slant and butt was observed to a purple .The two test may observed with or without gas and blackening on the media may be a presumptive positive results of Salmonella sp. Color on the Indole test gave a yellow ring formation, Methyl red test for diffuse red color of the media while Voges-Proskauer observed a colorless and Simmons Citrate appeared to be blue green with no growth as a positive results for Salmonella sp .However IMVIC Examination for Salmonella sp. on cooked burger patties was failed to indicates the specified color on the test, thus there was no Salmonella sp. that was being occurred from all the samples. (See appendix for raw data) In Table 2 showed the determined average pH of burger patties from 3 sampling period .pH 6.30 was the total average of all sample burger patties. pH is a measure of the acidity or basicity of an aqueous solution. Table 2 .The average pH of cooked burger patties from 3 sampling periods. Sampling period pH ST 1 Sampling period 6.27 2nd sampling period 6.31 rd 3 sampling period 6.34 Average= 6.30

26

Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline. In terms of acidity of meat products such as burger, their pH constantly was changing by the presence of pathogen reacting on the meat. The samples from the three sampling period in terms of their average pH are close to each other. The pH values of the samples ground beef patties ranged from 5.6 to 6.2 which is the normal values. The range of pH of the samples greater than 7 indicated the presence of the pathogen (Snyder et al., 1998).The first sampling period got the lowest pH which falls under the normal values. Sampling period two (2) and three (3) are slightly elevated which means that the samples were not contaminated or pathogenic to E. coli O157:H7 and Salmonella sp. Table 3 .Growth occurrence on total plate count of cooked burger patties from 8 randomly selected stalls.Stall numbe r N1 N2 N3 N4 E1 E2 E3 E4 1st sampling 2nd sampling 3rd sampling Total

4.9 x10-2 4.9 x10-2 1.83 x10-2 2.51 x10-2 3.3 x10-2 2.9 x10-1 6.4 x10-2 9.38 x10-2 4.96 x100

9.8 x10-2 1.5 x10-2 1.73 x100 1.1 x10-2 1.5 x10-2 3.8x10-3 3.4 x10-2 1.5 x10-2 1.92 x100

1.3x10-1 1.5x10-2 1.52 x100 1.1x10-2 1.5 x10-2 3.8x10-3 3.4 x10-2 1.5 x10-2 1.73 x100

2.77x10-1 7.9x10-2 5.08 x100 2.53 x100 6.3 x10-2 2.97 x10-1 1.32 x10-1 1.24 x10-1 *8.60 x100

* 8.60 x 10o (0.93 cfu /g), the total count of all bacteria present on cooked burger patties.

27

In Table 3, Total Plate count showed that there is a very low number of bacteria (in general) present on the cooked burger samples in 8 stalls in Polytechnic University of the Philippines North and East wing Canteen. From the 8 stalls of burger patties, it showed that E1 has the lowest total plate count which has 6.3 x10-2 and N3 has the highest total plate count of bacteria which has 5.08 x100.However it does not mean to say that the bacterial that have grown on the Plate Count Agar (PCA) is specifically a Escherichia coli O157:H7 and Salmonella sp. because PCA is used to determine the general level of microorganism on the samples. Moreover, considering the infective dose of E. coli O157:H7 from the samples that range from 1 to 100 colony-forming units and Salmonella sp. range from 15 to 20 colonies forming units, its detection at such concentration like 10 -1, 10-2, and 10-3 poses a significant health risk. There is no need to test the significant difference between the growth occurrences of Escherichia coli O157:H7 and Salmonella sp. in North and East Wing Canteen, because no Escherichia coli O157:H7 and Salmonella sp had been occurred for all the samples.

28

CHAPTER 5 SUMMARY, CONCLUSIONS, RECOMMENDATIONS

In this study, Escherichia coli O157:H7 and Salmonella sp. was not detected its occurrence on cooked burger patties sold in the morning from 8 randomly selected stalls at Polytechnic University of the Philippines (North and East Wing Canteen). The average pH of cooked burger patties from three sampling periods was 6.30 correspond from the normal pH values of the samples. The presence of the two bacteria examined, in which total bacterial count (general), 8.60 x 100 (0.93 cfu /g) was very low. There is no need to test the significant difference between the distributions of Escherichia coli O157:H7 and Salmonella sp. in North and East Wing Canteen, for there are no

Escherichia coli O157:H7 and Salmonella sp. had occurred for all the samples. Upon conducting this research, it was concluded that there is no occurrence of Escherichia coli O157:H7 and Salmonella sp. on cooked burger patties sold in the morning at Polytechnic University of the Philippines (North and East Wing Canteen) .Total Plate Count was very low which indicates that cooked burger patties were safe from contamination of Escherichia coli O157:H7 and Salmonella sp. There is no need to test the significant difference between the growth occurrences of Escherichia coli O157:H7 and Salmonella sp.

29

The researcher recommend that apart from cooked burger patties, other food served should undergo a microbial detection for the presence of E. coli O157:H7 and Salmonella sp. that needed to be addressed in order reduce public health risk posed by this organism. It was also recommended to assess other microorganisms like Staphylococcus aureus, Proteus sp. and coliform that were found in food industry as indicator organism. Moreover, it was also recommended to conduct bacterial examination in burger patties not only on the month of December, but also include the other season especially when numerous patties was sold by the stall owners. Consider also the time of sampling, the researcher, also patties sold in the afternoon. suggest for wider study to include testing the

30

LITERATURES CITED

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Collis, V. J., Reid, C.-A., Hutchison, M. L., Davies, M. H., Wheeler, K. P. A., Small, A., & Buncic, S. 2004. Hide Contamination with Marker Bacteria in Cattle in a simulated Livestock Market and an Abattoir. Journal of Food Protection. 67, 23972402. DAoust. J.Y., Maurer, J., Bailey, J.S. 2001. Salmonella species. In Food Microbiology: Fundamentals and Frontiers, 2nd Edition. ASM Press, Washington, DC. 141-178. Gorman, R., Bloomfield, S., & Adley, C. C. 2002. A study of Cross Contamination of Food-borne Pathogens in the Domestic kitchen in the Republic of Ireland. International Journal of Food Microbiology, 76, 143150 Larry Maturin and James T. Peeler. 1998. Bacteriological Analytical Manual, Edition 8, Revision A Karmali, M.A., Steele, B.T., Petric M., & Lim, C. 1983. Sporadic Cases of Haemolytic-Uremic Syndrome Associated with Faecal Cytotoxin-producing Escherichia coli in Stools. Lancet. 619-620. Kassenborg, H.D., Hedberg, C.W., & Hoekstra, M. 2004. Farm Visits and undercooked Hamburgers as Major Risk Factors from Sporadic Escherichia coli O157:H7 Infection: Data from a Case-control Study in 5 Food Net Sites. Clinical Infections. 38, S271-278. M. Sognorini, H. Tarabla. 2009. Quantitative Risk Assessment for Verocytotoxigenic Escherichia coli in Ground Beef Hamburgers in Argentina. International Journal of Food Microbiology. 87, 156150

Norval J. C. Strachan, Geoffrey M. Dunn, Mary E. Locking, Thomas M. S. Reid, Iain D. Ogden. 2006. Escherichia coli O157: Burger Bug or Environmental Pathogen? International Journal of Food Microbiology. 76, 143258 Neema Mrema, Sisai Mpuchane, Berhanu A. Gashe. 2006. Prevalence of Salmonella in Raw Minced Meat, Raw fresh Sausages, and Raw Burger Patties from retail outlets in Gaborone, Botswana. International Journal of Food Microbiology.76, 143258 Nyeleti, C., Hildebrandt, G., Kleer, J., & Molla, B. 2000. Prevalence of Salmonella in Ethiopian cattle and minced beef. Berl Munch Tierarztl Wochenschr. 113, 431-434. O. Peter Snyder, Jr. 1998. A Summary Of Research Information On Why Internal Meat Color Should Not be Used As an Indication of Meat Doneness. Hospitality Institute of Technology and Management.St. Paul. MN 55114

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Pan, Z., Singh, R. P., & Rumsey, T. R. 2000. Predictive Modelling of ContactHeating Process for Cooking a Hamburger Patty. Journal of Food Engineering, 46(1), 919 Paul S. Mead, Patricia M. Griffin. 1998. Escherichia coli O157:H7.Lancet Reij, M. W., Den Aantrekker, E. D., & ILSI Europe Risk Analysis in Microbiology Task Force. 2004. Recontamination as a source of pathogens in processed foods. International Journal of Food Microbiology. 91, 1-11. Robert V. Tauxe. 2002. Surveillance and investigation of Food Borne Diseases; Roles for Public Health in Meeting Objectives for Food Safety. S. K. Tamminga., R. R. Beumer, E.H Kampelmacher. 1982.Microbiological Study about Hamburger. Laboratory for Food Microbiology and Hygiene, agricultural University, Wageningnen, the Netherlands. USDA-FSIS. 1993. Heat processing cooking, cooling, handling and storage Requirements for uncured meat patties. Fed. Red. 58, 4113841152. Wachtel, M. R., Mcevoy, J. L., Luo, Y., William Campbell, A. M., & Solomon, M. B. 2003. Cross-contamination of lettuce by Escherichia coli O157:H7 via contaminated ground beef. Journal of Food Protection. 66, 11761183. Wells, S. J., Fedorka-Cray, p. J., Dargartz, D. A., Ferris, K., & Green, A. 2001. Faecal Shedding of Salmonella spp. by dairy cows on farm and at cull cow markets. Journal of Food Protection. 64, 311.

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APPENDICES

34

APPENDIX A Letter of Request to Conduct at National Meat Inspection Service (NMIS)

Polytechnic University of the Philippines College of Science Department of Biology _________________________________________________August 9, 2010 Dr. Pedro Jerry D. Baliang Executive Director National Meat Inspection Service Central Office BAI Compound Visayas Avenue, Diliman, Quezon City Dear Sir: Good day. We the 4th year students of Bachelor of Science in Biology of Polytechnic University of the Philippines. We are currently enrolled in the subject Research I (Thesis Proposal) this 1st semester SY 2010-2011 as a requirement of our course. The proposed title of our research is Detection on Selected Pathogenic Microorganisms on Burger Patties sold at Polytechnic University of the Philippines Canteen (North and East Wing). We will be conducting the experimental part of our research in the second semester SY 2010-2011 to complete our subject Research II (Experimental). Although we have laboratory facilities in our University, we dont have the enough facilities to conduct our experiment. In view of this, we would like to request for your authority to allow us to conduct the laboratory part of our experiment at NMIS. As one of the thrust of your institution, we believe that the result of this experiment could be very helpful to keep s aware that burger patties sold at Polytechnic University of the Philippines are free from pathogenic microorganisms which could be possible causes of disease to people. We are hoping for your kind heart to favorably grant our request. Thank you very much.. Respectfully yours, ALVIN B. BUENCONSEJO MILAGROSA C. MANGOMA BS BIOLOGY 4-1D

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APPENDIX B Preparation of Media Buffered Peptone Water (BPW) Buffered Peptone Water (BPW) is rich in nutrients and used for the preliminary selective of bacteria, particularly pathogenic microorganism (Enterobacteriacea) from food stuff. Procedure 1. Weigh 12.75 grams of Buffered Peptone Water (BPW) and transfer into Erlenmeyer flak 2. Add 500 ml distilled water and cover with aluminum foil. Heat with frequent agitation and boil for 1 minute to dissolve using hot plate with magnetic stirrer inside the flask. 3. Sterilize in autoclave into 121o C for 15 minutes at 15 psi. 4. Cool to 45- 50o C and dispense 225 ml into sampling bottle .These are now ready to use. 5. If the broth is not to be used immediately, pack the sampling bottle in autoclave plastic bags and store in refrigerator at 2 o to 4o C Tryptic Soy Broth with Novoniocin (TSB + Novobiocin) Tryptic Soy Broth with Novoniocin (TSB + Novobiocin) is use for selective enrichment of enterohemorrhagic E. coli O157:H7. Procedure: 1. Weigh 33 g of Typtic Soy Broth with Novoniocin ( TSB + Novobiocin ) and transfer into Erlenmeyer flask 2. Add 1000 ml distilled water .Cover the flask with aluminum foi 3. Dissolve and stir using hot plate with magnetic stirrer inside the flask. 4. Sterilize in autoclave into 121o C for 15 minutes at 15 psi 5. Cool to 45- 50o C and pour 10 15 ml of agar into sterile Petri dishes carefully to avoid bubbles 6. Partially cover the plates to avoid contamination. Cool until agar hardens and completely cover the plates .These are now ready for use. 7. If the agars are not to be used immediately, pack in autoclave plastic bags and store in refrigerator at 2 o to 4 C

36

Plate Count Agar (PCA) Plate count agar is a microbiological growth medium commonly used to assess or to monitor "total" or viable bacterial growth of a sample. PCA is not a selective medium. Procedure 1. Weigh 27.5 g Plate Count Agar and transfer into Erlenmeyer flask. 2. Add 500 ml distilled water and cover with aluminum foil. 3. Heat with frequent agitation and boil for 1 minute to dissolve using hot plate with magnetic stirrer inside the flask. 4. Sterilize in autoclave into 121o C for 15 minutes at 15 p 5. Cool to 45- 50o C and pour 10 15 ml of agar into sterile Petri dishes carefully to avoid bubbles. Partially cover the plates to avoid contamination 6. Cool until agar hardens and completely cover the plates .These are now ready for use 7. If the agars are not to be used immediately, pack in autoclave plastic bags and store in refrigerator at 2 o to 4o C. Flourocult Agar Flourocult Agar is a selective media specifically for isolation and identification of E. coli O157:H7 Procedure 1. 2. 3. 4. 5. Weigh 27.5 g of Flourocult O157:H7 and transfer into Erlenmeyer flask. Add 500 ml distilled water and cover with aluminum foil. Dissolve and stir using hot plate with magnetic stirrer inside the flask Sterilize in autoclave into 121o C for 15 minutes at 15 psi. Cool to 45- 50o C and pour 10 15 ml of agar into sterile Petri dishes carefully to avoid bubbles. Partially cover the plates to avoid contamination 6. Cool until agar hardens and completely cover the plates .These are now ready for use. 7. If the agars are not to be used immediately, pack in autoclave plastic bags and store in refrigerator at 2 o to 4o C.

37

Tetrathionate Broth (TT Broth) Tetrathionate Broth (TT Broth) used as an enrichment broth for isolation of Salmonellae Procedure 1. weigh 17.5 g of Tetrathionate Broth ( TT Broth ) and transfer into Erlenmeyer flask 2. Add 500 ml distilled water and cover with aluminum foil. Heat with frequent agitation and boil for 1 minute to dissolve using hot plate with magnetic stirrer inside the flask. 3. dispense 10 ml into testube with cap.Cool the broth and these are now ready to use 4. If the broths are not to be used immediately, pack in autoclave plastic bags and store in refrigerator at 2 o to 4o C. Rappaport and Vassiliadis (RVS) Broth Rappaport and Vassiliadis (RVS) Broth is used for the selective enrichment of Salmonella with the exception of Salmonella typhimuruim. Procedure: 1. Weigh 32.7 g of Rappaport and Vassiliadis (RVS) and transfer into Erlenmeyer flask. 2. Add 1000 ml distilled water and cover with aluminum foil. Heat with frequent agitation and boil for 1 minute to dissolve using hot plate with magnetic stirrer inside the flask. 3. Dispense 10 ml into test tube with cap and sterilize in autoclave into 121 o C for 15 minutes at 15 psi 4. Cool the broth .These are now ready for use 5. If the broths are not to be used immediately, pack in autoclave plastic bags and store in refrigerator at 2 o to 4o C.

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Brilliant-green Phenol-red Lactose Sucrose Agar (BPLS) Brilliant-green Phenol-red Lactose Sucrose Agar (BPLS) a selective agar for the isolation and differentiation of Salmonella typhi and other Salmonellae. Procedure: 1. Weigh 23.75 g of Brilliant-green Phenol-red Lactose Sucrose Agar (BPLS) and transfer into Erlenmeyer flask 2. Add 500 ml distilled water and cover with aluminum foil. Heat with frequent agitation and boil for 1 minute to dissolve using hot plate with magnetic stirrer inside the flask. 3. Cool to 45- 50o C and pour 10 15 ml of agar into sterile Petri dishes carefully to avoid bubbles. Partially cover the plates to avoid contamination 4. Cool until agar hardens and completely cover the plates .These are now ready for use 5. If the agars are not to be used immediately, pack in autoclave plastic bags and store in refrigerator at 2 o to 4o C. Xylose Lysine Deoxycholate(XLD) Xylose Lysine Deoxycholate(XLD) is a selective agar for the isolation and differentiation of Salmonella sp. Procedure 1. Weigh 23.75 g Xylose Lysine Deoxycholate(XLD) and transfer into Erlenmeyer flask 2. Add 500 ml distilled water and cover with aluminum foil. Heat with frequent agitation and boil for 1 minute to dissolve using hot plate with magnetic stirrer inside the flask. 3. Cool to 45- 50o C and pour 10 15 ml of agar into sterile Petri dishes carefully to avoid bubbles. Partially cover the plates to avoid contamination 4. Cool until agar hardens and completely cover the plates .These are now ready for use 5. If the agars are not to be used immediately, pack in autoclave plastic bags and store in refrigerator at 2 o to 4o C.

39

Hektoen Enteric Agar (HEA) Hektoen Enteric Agar (HEA) is a selective agar for the isolation of Salmonella and Shigella. Procedure 1. weigh 37.5 g of Hektoen Enteric Agar (HEA) and transfer into erlemeyer flask 2. Add 500 ml distilled water and cover with aluminum foil. Heat with frequent agitation and boil for 1 minute to dissolve using hot plate with magnetic stirrer inside the flask. 3. Cool to 45- 50o C and pour 10 15 ml of agar into sterile Petri dishes carefully to avoid bubbles. Partially cover the plates to avoid contamination 4. Cool until agar hardens and completely cover the plates .These are now ready for use 5. If the agars are not to be used immediately, pack in autoclave plastic bags and store in refrigerator at 2 o to 4o C. Preparation of Biochemical Test a. Tryptone Sugar Iron (TSI) Procedure 1. Weigh 16 of Tryptone Sugar Iron and transfer into Erlenmeyer flask. 2. Add 500ml distilled water and cover the flask . Heat with frequent agitation and boil for 1 minute to dissolve using hot plate with magnetic stirrer inside the flask. 3. Dispense 2.5 ml into test tubes and sterilize in autoclave in 121o C for 15 minutes at 15 psi. 4. Slant the agar and cool until hardens. These are now ready to use. 5. If the agars are not to be used immediately, pack in autoclave plastic bags and store in refrigerator at 2 o to 4o C. b. Lysine Iron Agar Procedure 1. Weigh 16 g of Lysine Iron Agar (LIA) and transfer into Erlenmeyer flask 2. Add 500ml distilled water and cover the flask. Heat with frequent agitation and boil for 1 minute to dissolve using hot plate with magnetic stirrer inside the flask.

40

Dispense 2.5 ml into test tubes and sterilize in autoclave in 121 o C for 15 minutes at 15 psi 4. Slant the agar and cool until hardens. These are now ready to use 5. If the agars are not to be used immediately, pack in autoclave plastic bags and store in refrigerator at 2 o to 4o C 3. c. Indole Preparation 1. weigh 12.12 grams of Indole and transfer into Erlenmeyer flask 2. Add 500 ml distilled water and cover the flask. Heat with frequent agitation and boil for 1 minute to dissolve using hot plate with magnetic stirrer inside the flask. 3. Dispense 2.5 ml into test tubes with cap and sterilize in autoclave in 121o C for 15 minutes at 15 psi 4. Cool the medium. These are now ready to use 5. If the meduim are not to be used immediately, pack in autoclave plastic bags and store in refrigerator at 2 o to 4o C

d. Methyl red (MR) and Voges-Proskauer (VP) Procedure 1. Weigh 8.5 g of Methyl red (MR) and Voges-Proskauer (VP) and transfer into Erlenmeyer flask 2. Add 500 ml of distilled water and cover the flask with aluminum foil. Heat with frequent agitation and boil for 1 minute to dissolve using hot plate with magnetic stirrer inside the flask. 3. Dispense 2. 5 ml into test tube with cap and sterilize in autoclave in 121 C for 15 minutes at 15 psi. 4. Cool the medium .These is now ready to use 5. If the medium are not to be used immediately, pack in autoclave plastic bag and store in refrigerator at 2 to 4 C

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e. Simmons Citrate (SC) Procedure 1. Weigh 11. 25 g of Simmons Citrate ( SC) and transfer into Erlenmeyer flask 2. Add 500 ml distilled water and cover with aluminum foil . Heat with frequent agitation and boil for 1 minute to dissolve using hot plate with magnetic stirrer inside the flask. 3. Dispense 2.5 ml into test tubes with cap and sterilize in autoclave in 121o C for 15 minutes at 15 psi 4. Slant the agar and cool until hardens. These are now ready to use 5. If the agars are not to be used immediately, pack in autoclave plastic bags and store in refrigerator at 2 o to 4o C

42

APPENDIX C Summary of Presumptive Positive and Confirmation Results of E. coli O157:H7Table 1 shows the presumptive positive reaction on color colony, UV Light reaction on the medium used and the confirmation of the E. coli O157:H7 Meduim Flourocult Color colony Colorless Ultra Violet Light reaction Not fluorescence Confirmatory test Agglutination /precipitate occur

Summary of Presumptive Positive and Confirmation Results of Salmonella sp. a. Salmonella sp. colony colorMedia Color colony

Hektoen Enteric Agar (HEA) Xylose Lysine Deoxycholate (XLD) Brilliant-green Phenol-red Lactose Sucrose (BPLS)

Blue green colony Pink colony with or without black center. Pink colony

Table 2 shows the presumptive positive colony color of Salmonella sp on the media.

b.Biochemical Testportion slant butt Tryptone Sugar Iron (TSI) Red slant Yellow butt Lysine Iron Agar ( LIA) Purple slant Purple butt Gas formation Bubles or gas formation occur Bubles or gas formation occur Blackening ( H2S) + +

Table 3 shows the presumptive positive results on TSI, LIA, gas formation, and the presence of blackening on the media.

IMVIC Examination Results for Salmonella sp.Indole Yellow ring formation Methyl red Brick red Voges-Proskauer No color Simmons citrate Blue green and no growth

Table 4 shows the confirmation results of positive Salmonella sp. on IMVIC examination.

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APPENDIX D Raw Data for E. Coli O157:H7 1st Sampling for E. Coli O157:H7 on Cooked Burger Patties from 8 stalls Legend : Stall Color of colony *Ultra violet light **Confirmatory ( Fluorocult) reaction test N1a Black with Negative Negative yellow N1b colorless Negative Negative N1c colorless Negative Negative N2a yellow Negative Negative N2b Black and Negative Negative yellow N2c yellow Negative Negative N3a yellow Negative Negative N3b yellow Negative Negative N3c colorless Negative Negative N4a yellow Negative Negative N4b yellow Negative Negative N4c yellow Negative Negative E1a Yellow with Negative Negative black E1b yellow Negative Negative E1c yellow Negative Negative E2a Black and Negative Negative yellow E2b yellow Negative Negative E2c yellow Negative Negative E3a yellow Negative Negative E3b Black and Negative Negative yellow E3c yellow Negative Negative E4a yellow Negative Negative E4b Black and Negative Negative yellow E4c yellow Negative Negative *Negative - does not fluorecence when subjected to ultraviolet rays Remarks Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

** Negative does not precipitate when E.coli O157:H7 reagent was added and mix to the colony. Black and yellow colony negative E.coli O157:H7 Colorless colony Presumptive positive for E.coli O157:H7

44

2nd Sampling for E. coli O157:H7 on Cooked Burger Patties from 8 stalls Stall Color of colony ( Fluorocult) yellow Black yellow yellow Black with yellow yellow yellow yellow yellow yellow yellow yellow yellow yellow yellow Black with yellow yellow yellow yellow yellow yellow yellow yellow yellow Ultra violet light reaction Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Confirmatory test Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Remarks

N1a N1b N1c N2a N2b N2c N3a N3b N3c N4a N4b N4c E1a E1b E1c E2a E2b E2c E3a E3b E3c E4a E4b E4cLegend :

Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

*Negative - does not fluorecence when subjected to ultraviolet rays ** Negative does not precipitate when E.coli O157:H7 reagent was added and mix to the colony. Black and yellow colony negative E.coli O157:H7 Colorless colony Presumptive positive for E.coli O157:H7

45

3rd Sampling for E. coli O157:H7 on Cooked Burger Patties from 8 stalls Stall Color of colony ( Fluorocult) yellow Black yellow yellow yellow yellow yellow yellow Colorless yellow yellow yellow yellow colorless yellow yellow yellow yellow yellow colorless yellow yellow yellow yellow Ultra violet light reaction Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Confirmatory test Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Remarks

N1a N1b N1c N2a N2b N2c N3a N3b N3c N4a N4b N4c E1a E1b E1c E2a E2b E2c E3a E3b E3c E4a E4b E4cLegend:

Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

*Negative - does not fluorecence when subjected to ultraviolet rays ** Negative does not precipitate when E.coli O157:H7 reagent was added and mix to the colony. Black and yellow colony negative E.coli O157:H7 Colorless colony Presumptive positive for E.coli O157:H7

46

APPENDIX ERaw Data for Salmonella sp. On Cooked Burger Patties

First Sampling Raw Data for Salmonella sp. On Cooked Burger PattiesLegend : Total Plate Count Stalls N1a N1b N1c N2a N2b N2c N3a N3b N3c N4a N4b N4c E1a E1b E1c E2a E2b E2c E3a E3b E3c E4a E4b E4c Weight (g) 37.13 38.26 39.38 33.92 36.02 39.01 33 34.76 35.72 37.92 35.92 35.38 38.93 38.56 35.67 32.7 31.31 34.11 36.4 35.91 38.83 29.43 33.76 30.8 pH 6.36 6.46 6.4 6.35 6.47 6.52 6.27 5.69 5.5 6.3 6.15 6.2 6.44 6.61 6.15 6.74 6.44 6.46 6.36 6.18 6.64 6.18 6.32 6.36 12 5 0 0 5 0 0 1 55 28 50 0 44 2 0 0 47 2 13 4 0 9 0 9 0 4 0 2 1 0 1 9 170 29 59 2 165 0 0 2 5 1 2 1 0 1 5 3 2 13 1 0 1 2 0 4 2 194 0 44 19 268 0 0 5 6 2 1 1 0 1 0 4 2 14 tota l 10 0 3 8 0 5 12 419 57 153 21 474 2 0 7 58 5 16 6 0 12 5 16 4 Bacterial Isolation BPLS T+, R+ T+ T+ T+ HEA XLD -

TSI Slant R butt Y H2S + Gas Slant P

LIA Butt P H2 S +

Biochemical test I M + VP CS + Remarks Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

R

Y

-

+

P

P

+

-

+

-

+

Negative Negative Negative Negative Negative Negative Negative Negative Negative

R Y + P P + + R Y + P P + + +

Negative Negative Negative

R- red color Y- yellow color O- orange color P- purple color

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Second Sampling Raw Data for Salmonella sp. On Cooked Burger Patties

Total Plate Count Stalls N1a N1b N1c N2a N2b N2c N3a N3b N3c N4a N4b N4c E1a E1b E1c E2a E2b E2c E3a E3b E3c E4a E4b E4c Weight (g) 37.24 38.24 34.63 36.94 39.5 36.08 37.4 34.84 35.84 35.83 31.16 32.26 37.14 39.61 39.21 34.88 32.73 35.39 35.48 32.69 39.68 28.94 38.3 32.01 pH 6.58 6.54 6.47 6.47 6.12 6.25 5.87 6.1 5.86 6.15 6.18 6.24 6.4 6.44 6.4 6.44 6.3 6.38 6.3 6.34 6.29 6.27 6.38 6.26 12 10 1 0 0 0 2 56 0 198 0 2 1 1 1 0 0 1 0 2 2 0 2 0 0 13 8 1 1 0 0 2 10 2 173 0 0 0 0 2 0 0 0 0 1 1 2 0 0 0 14 2 1 2 0 0 0 7 1 12 0 0 0 0 0 0 0 0 0 0 0 1 2 0 0 tota l 20 3 3 0 0 4 73 3 383 0 2 1 1 3 0 0 1 0 3 3 3 4 0 0

Bacterial Isolation BPLS T+ T+ T+ T+ T+ T+ T+ T+ HEA XLD T+

TSI Slant Y R butt O Y H2S Gas + +

LIA Slant P P Butt Y Y H2 S -

Biochemical test I M VP CS Remarks Negative Negative Negative

R Y Y

Y Y Y

-

+ -

P P P

Y Y Y

+ -

Negative Negative Negative Negative

R R

0 O

-

-

P

Y

-

Negative Negative Negative

R

O

-

+

P

Y

-

Negative Negative Negative Negative Negative Negative Negative Negative Negative

Y

O

-

+

P

Y

-

Negative Negative Negative Negative Negative

-

Legend : R- red color Y- yellow color O- orange color P- purple color

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Third Sampling Raw Data for Salmonella sp. On Cooked Burger PattiesTotal Plate Count Stalls N1a N1b N1c N2a N2b N2c N3a N3b N3c N4a N4b N4c E1a E1b E1c E2a E2b E2c E3a E3b E3c E4a E4b E4c Weight (g) 37.24 38.24 34.63 36.94 36.08 39.5 37.4 34.84 36.08 39.61 32.26 37.14 31.16 34.88 39.21 35.48 32.73 35.39 28.94 38.3 39.68 34.49 32.69 38.94 pH 6.58 6.13 6.31 5.87 6.25 6.04 5.81 6.18 5.86 6.15 6.18 6.24 6.22 5.91 6.14 6.38 6.3 6.3 6.29 6.23 6.34 5.88 6.27 6.17 12 5 1 0 0 0 2 48 0 175 0 2 1 1 1 0 0 1 0 1 0 0 2 0 0 13 9 1 2 0 0 2 8 2 156 0 0 0 0 2 0 0 0 0 2 2 2 0 0 0 14 14 1 1 0 0 0 7 1 7 0 0 0 0 0 0 0 0 0 0 1 1 2 0 0 tota l 28 3 3 0 0 4 63 3 338 0 2 1 1 3 0 0 1 0 3 3 3 4 0 0 Bacterial Isolation BPLS T+ T+ HEA XLD -

TSI Slant butt H2 S Gas

LIA Slant Butt H2S

Biochemical test I M VP CS Remarks Negative

R

Y

-

+

P

Y

-

Negative Negative Negative

Y

Y

-

+

P

Y

-

Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

-

Legend : R- red color Y- yellow color O- orange color P- purple color