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TITLE PAGE

ENUMERATION OF BACILLUS CEREUS FROM FOOD SAMPLE SOLD IN PRIMARY AND SECONDARY SCHOOL, ILORIN METROPOLIS

BY

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EE/H2013/01430
DEPARTMENT OF ----
SCHOOL OF ---
INSTITUTE OF ---

DECEMBER,2018

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APPROVAL PAGE

This is to certify that the research work, "enumeration of bacillus cereus from food sample sold in primary and secondary school, ilorin metropolis" by ---, Reg. No. EE/H2007/01430 submitted in partial fulfillment of the requirement award of a Higher National Diploma on --- has been approved.

By
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Supervisor                                                  Head of Department.
Signature……………….                           Signature……………….        

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External Invigilator

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DEDICATION
This project is dedicated to Almighty God for his protection, kindness, strength over my life throughout the period and also to my --- for his financial support and moral care towards me.Also to my mentor --- for her academic advice she often gives to me. May Almighty God shield them from the peril of this world and bless their entire endeavour Amen.

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ACKNOWLEDGEMENT

The successful completion of this project work could not have been a reality without the encouragement of my --- and other people. My immensely appreciation goes to my humble and able supervisor mr. --- for his kindness in supervising this project.
My warmest gratitude goes to my parents for their moral, spiritual and financial support throughout my study in this institution.
My appreciation goes to some of my lecturers among whom are Mr. ---, and Dr. ---. I also recognize the support of some of the staff of --- among whom are: The General Manager, Deputy General manager, the internal Auditor Mr. --- and the ---. Finally, my appreciation goes to my elder sister ---, my lovely friends mercy ---, ---, --- and many others who were quite helpful.

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TABLE OF CONTENT
Title page                                                                 
Certification                                                                       
Acknowledgement                                                             
Table of content                                                                
Abstract                           
                                                 
CHAPTER ONE                                                                
1.0    Introduction and Literature Review
1.1    Aims and Objective

CHAPTER TWO                                                                       
2.0    Sterilization of Equipment                                           
2.1    Sample Collection                                                       
2.3    Laboratory Preparation of                                           
2.4    Processing of Sample                                                 
2.5    Enumeration of Bacillus Cereus from                            
2.6    Food Sample using Most Probable Number (MPN)         
2.7    Culturing of Bacillus Cereus          
                                       
CHAPTER THREE
3.0    Result analysis

CHAPTER FOUR
4.0    Discussion
4.1    Recommendation
4.2    Conclusion
4.3    References 

CHAPTER ONE
1.0   INTRODUCTION AND LITERATURE REVIEW
        B. Cereus is a spore forming organism capable of developing at a whole range of temperature, PH and water activity values. (Barker et al; 2005) thus bacterium, frequency associated with food born disease can be found in the natural environment and isolated from various foods, including meat and meat production (Ahmed et al; 1983).
Some of the enterotoxins produced by B. Cereus causes food born diarrhea, characterized as the “diarrheic syndrome” this syndrome can be associated with the productin of five different protein, haemolysine, enterotoxin and enteroxins, non haemolytic enteroxine cytotoxins, the symptoms last for a period of 12 to 24 hours and consist of abnormal pain, watery diarrhoea and nausea. It is believed that the enterotoxins are mostly produced in the small intestine of the individual (Ankolekar et al; 2009) food associated with outbreaks of B cereus diarrhoea fr4equency include protein rich foods and researcher’s have emphasized the importance of the presence of microorganism or its spores in meat and meat products for the occurrence of such outbreaks practices such as inadequate temperature during food processing are indicated as the main factors contributing to the spread of the microorganism in these foods, with the objective of investigating the growth of enterotoxin producing B. Cereus in processed meat, this study was carried out using cultures of the microorganism from different origin (Bernhard et al; 1978) it is frequently isolated from milk and dairy products (Ahmed et al 1983)  in milk B. Cereus causes a defect known as “bitty” cream or sweet curding. It is found in rice, rice products oriental dishes and ingredients (Blaky et al; 1980) a variety of foods have been implicated in food-poisoning (Johnson, 1984) frequently isolated from soil and growing plants in the intestinal track of insect and mammals (Arnesen et al; 2008)
Germination of most of the enterotoxigenic strain of B. Cereus adhered to CaC0-2 cells is stimulated while another celline HEP-2 does not trigger germination (Wijnands et al, 2007). The germination stimulated by Cac0-2 cells is related to the normal functions of germination receptors (Horustra et al, 2009) Beta-lactamase type I occur in the sporalated for run in penicillin-resistant B Cereus (Fenselau et al, 2008)
B. Cereus is easily isolated and identified from samples clinical specimens and food samples are collected, stored temporary at refrigerating temperature and plated on selective agar media (Boschioits et al, 1983) low level of peptone and the absence of carbohydrates in KG medium facilitated formation of spore (Johnson, 1984). A columbia base 5% blood agar surface-spread with polymyxin B is recommended by Kramer et al. (Kramer et l, 1982) for better colonly characteristic and colony types are easily differentiated for serological testing in enumeration by most probable number method, trypticase soy polymyxin broth is recommended.
Bacaragar is a modified selective agar and also contains Bacara and egg yolk. Pyruuate is found in rice, rice products, oriental dishes and ingredients (Blakey et. Al. 1980; et al, 1986). A variety of foods have been implicated in food-poisoning.
Emetic syndrome caused by bacillus cereus is highly associated with rice and rice products (Johnson, 1984).
Reauire both Gerl and area germination receptors for germination in inosine as the sole germinant, whereas the Gerl receptor is responsible for most of the response to L-alarine as the sole germinant, with a smaller contribution from identify with its homolog Gern which is a Na+/H+-K+ antiporter that is required for normal spore germination, while it has a significant role in outgrowth; gert mutant spores do not outgrow efficiently under alkaline conditions and outgrow more slowly than the wild type in the presence of high Nacl concentrations. The Gert protein in B. Cereus therefore countibutes to the success of spore outgrowth from the germinated state during alkaline or Na+ stress (senior et al, 2008).
Germination of most of the enterotoxigenic strains of B. cereus adhered to caco-2 cells is stimulated while another celline, Hep-2, does not trigger germination (wijnards et al; 2007). The germination stimulated receptors (Hornstra et al. 2009).
Beta-lactamase type I occurs in the sporulated from the in penicillin resistant B. cereus (fenselau et alp; 2008) B. cereus is easily isolated and identified from samples chemical; specimens and food samples are collected, stored temporary at refrigerating temperature if necessary, diluted if necessary, and plated on selective agar media.
Mami egg yolk polymyxin agar (MYP) is usually recommended, polymyxin is the selective agent, and egg yolk and mannitol are differential agents. Typical colonies are rough with.
B. Cereus is differentiated from other non motile species by forming diffuse growth on predried nutrient agar as B. Cereus Var. mycoides B.cereus is usually strong B-hemolytic on trypticase soy sheep blood agar plate, whereas, B. thuringiensis and B. cereusvar. Mycoides are often weakly hemolytic and B. anthracis is non motile and non hemolytic. Endotoxcin crystals are found in B. thuringiensis (Harmon, 1978). Other biochemical tests used in confirming B. cereus are: acid is produced from anaerobic glucose fermentation, nitrate reduction, VP positive, tyrosine, decomposition, selective growth on blood agar and bacara agar, Kramer etal, 1982) recommended the following biochemical test for confirmation of B. cereus: Glucose +, mannotol, Xyclose-. And arabinose-, serotyping is recommended by a number of investigators (Bernhard et al 1978).
B. anthracis and other Bacillus species including B.cereus can also be identified by the rapid apitests with the aid of computer programmes (Logan et al., 1985).
Among those strains of B. cereus isolated from different foods and environmental samples, predominance of small cluster of serotypes characteristics of the source is apparent, (Gilbert et al., 1982)
Nonhaemolytic enterotoxin and cytotoxin k are currently considered the aetiological agents of B. cereus related diarrhoeal food borne disease. Hbi and Nhe are related three component toxins, while the single component cytk belongs to the family of b-barrel pore-forming toxin. In addition, several other protein cy totoxins, haemolysis and degraduative enzymes have been described that may potentially contribute to the pathogenicity of B, cereus diarrhoeal disease. These include cereolysin O, haemolysin TT haemolysin III and three phosphotipases C (Stenfors et al, 2008)
CHARACTERISTIC OF THE TWO TYPES OF BACILLUS COREUS FOODBORNE DISEASE

Characteristics	Diarrhoeal disease	Emetic disease
Types of Toxin	Protein: Enterrotoxin(S) Hbl, Nhe, Cytk implicated	Cyclicpeptide: emetic toxin (cereulides)
Location of Toxin	In the small intestine of the host.	performed in foods
Infective Dose	105-106CFU (total) the total number required is lower for spores compared to vegetative cells	105-106 cells is often found in implicated foods but live cells are not required for intoxication ceretice 8-10kg-7 weight caromal mode(s)
Incubahon Time	8-16h (occasionally 24h)	5-6h
Duration of Illness	12-24h (occasionally several days)	6-24h
Symptoms	Abdominal pain, watery diarrhoea and occasionally nausea lethality has occurred	Nausea, Vomiting and Malaise. A few lethal cases *(Possibly due to liver damage)
Food most Frequently Implicated	Protein aceous foods, meat products, so ups, vegetables, puddings, Sauces, milk and milk product	Starch rich foods, fried and cooked rice, pasta and noodles.

 
(Stenfors et al, 2008)
1.1   AIMS AND OBJECTIVES
The main objective of this project is to;

• Determinacy the highest frequently of bacillus cereus occurrence in selected food samples.
• Formulation of a provincial food monitoring system.
• Proper isolation and control
• Education of food handler/sellers and analysis in food safely.
• Give strategic directives for the development of food control and food safety.
• Outline the nature of food monitoring which are needed in the medium
• Identify the facilities, resources and staff required for rendering the monitoring
• Promotion of the management system for food safety assurance.
• To find an effective cure to the food-borne disease
• To know its occurrence in food and management.

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