TITILE PAGE

ISOLATION AND IDENTIFICATION OF BACTERIA FROM FOOD VENDORS

AND SOME VEGETABLE AVAILABLE AT OGBETE MARKET ENUGU.
BY
EDEH ANASTHECIA N.
MB/ 2008/385
A RESEARCH PROJECT (MCB429) SUBMITTED IN PARTIAL FULFILLMENT
FOR THE AWARD OF BACHELOR OF SCIENCE (B.SC) DEGREE IN
MICROBIOLOGY AND BIOTECHNOLOGY
IN THE DEPARTMENT OF MICROBIOLOGY AND BIOTECHNOLOGY
FACULTY OF NATURAL SCIENCES CARITAS UNIVERSITY AMORJI NIKE
ENUGU
SUPERVISOR
PROF BRYAN OGENEH
AUGUST, 2012
CERTIFICATION PAGE
I certify that this research project was carried out by Edeh Anasthecia. N. (MB/ 2008 (385)
in the Department of Microbiology and Biotechnology, faculty of Natural Sciences,
Caritas university. Amorji Nike Enugu. The Department recognizes that Edeh Anasthecia
N. (MB/2008/385) bears full responsibility for this work.
………………………. …………………………...
Edeh Anasthecia N. Date
Student
………………………… …………………………
Prof Bryan Ogeneh Date
Supervisor
……………………………… …………………………….
Nnema E.E Date
HOD
……………………………… ………………………………
External Examiner Date
DEDICATION
This work is dedicated to Almighty God and my mother Mary, for protection and love
throughout my four years in school and to my parent (Mr. & Mrs. Simeon Edeh) for
considering my education a priority.
ACKNOWLEDGEMENT
A Marvelous thanks to almighty God for his wisdom, protection, favour, and blessing.
And to my incomparable and treasured parent Mr. & Mrs. S.O.N Edeh who made my
dream come true, also to my siblings Johnbosoco, Priscilla, Irene, Paul and Cynthia Edeh
for their prayers and to my aunty Mrs. Rosemary Israel my friend Akueme Maximillian. I
owe you all a lot.
I also wish to express my sincere gratitude to my supervisor Prof. Bryan Ogeneh for the
support and time he dedicated for my work, my dean, HOD and to all my lecturers in the
department of microbiology and Biotechnology I will never forget you all.
Finally to my friends, room mates and course mates who in one way or the other
contributed to my work I say thanks and God bless.
TABLE OF CONTENTS
Title page ……………………………………………………… i
Certificate page………………………………………………… ii
Dedication …………………………………………………….. iii
Acknowledgement ……………………………………………. iv
Table of contents ……………………………………………… v
List of tables …………………………………………………... vii
Abstract ……………………………………………………….. viii
Chapter One
1.0 Introduction……………………………………………… 1
1.1 Aim and Objectives …………………………………….. 6

Chapter Two
2.1 Literature review……………………………………………. 7
2.2 Food hygiene………………………………………………... 12
2.3 Factors that contribute to food borne illness……………….. 13
2.4 Features of common food –borne pathogens ……………… 16
2.5 Food- born diseases ………………………………………. 25
2.6 Pre-disposing factors to food-born illness ………………… 26
2.7 Prevention of food born illness……………………………. 27
Chapter Three
3.1 Materials and method……………………………………. 29
3.1.1 Hard ware ……………………………………………… 29
3.1.2 Soft ware……………………………………………….. 29
3.2 Methods …………………………………………………. 31
3.3 Identification of isolates ………………………………… 33
Chapter Four
4.1 Results ………………………………………………….. 40
Chapter Five
5.1 Discussion……………………………………………….. 46
5.2 Conclusion ……………………………………………… 48
5.3 Recommendation……………………………………….. 49
References
Appendix
LIST OF TABLES
i. some food borne bacteria and affiliated foods ………………22
ii. Morphological features of bacteria isolated from the different food
samples…………………………………………………40
iii. Total bacterial count (CFU/mg) of street vended foods
samplers…………………………………………………….43
iv. Biochemical test……………………………………………44
v. Food samples with their associated…………………………45
ABSTRACT
Safety of food is a basic requirement of food quality. A total of 25 street food samples
(Jollof rice, egwusi soup, ugu, water leaf and green), were randomly purchased from five
different vendors in Ogbete main market Enugu. The samples were transported in ice to
the laboratory. The samples were bacteriologically analyzed using pour plate technique
and sub-culture. Pour plate techniques was done by carrying out serial dilution of the
sample after which the first tube and the last tube were picked and 1ml of each sample was
pipette into a Nutrient agar, the plate was then incubated for 24 hours at 37 oC after which
the plate were examined for growth. Sub culture was done using bacteriological agar. All
the screened food samples had varying levels of bacterial growth ranging from 1.0 X 105 to
3.0 X 106 cfu/ml. ninety percent of the sampled foods had bacterial counts above the
acceptable limits (104 cfu/ml) and 10% of the samples had bacterial counts less than (<104
cfu/ml). Six bacterial species were isolated from the foods sampled. Staphylococcus
arueus, Bacillus cereus, Vibrio spp, Salmonella spp, Escherichia coli and Shigella spp.
More than one pathogenic micro organism were isolated from jollof rice and water leaf.
The findings revealed that street foods are potential vehicles for transmitting food borne
illnesses thus the need to develop practical strategies geared toward street food safety.
CHAPTER ONE
1.0 INTRODUCTION

Bacterial are group of microorganism all of which lack a distinct nuclear membrane (and
hence are considered more primitive than animal and plant cells) and most of which have a
cell wall of unique composition. Most bacterial are unicellular; the cells may be spherical
(coccu) rod – shaped (bacillus), spiral (spirillum), comma – shaped (vibrio) or corkscrew-
shaped (spierocheate). Generally, they range in size between 0.5 and 5um. (Elizabeth and
Martin, 2003).
Food is any substance that people or animal eat or drink or that plants absorb to maintain
life and growth. Food is any substance consumed for nutritional support for the body; it is
usually of plant or animal origin. (Ezeronye, 2007). Food consists of chemical compounds
which heterophilic living thing consumes in order to carry out metabolic processed. They
are also substances which when introduced to the digestive system under normal
circumstances contribute to growth, repair and production of energy.
(Ezeronye, 2007). Foods are classified into six essential nutrients known as protein,
carbohydrate, vitamin mineral, fat and oil, water.
PROTEIN: - One of a group of organic compounds of carbon, hydrogen, oxygen and
nitrogen (sulphur and phosphorus may also be present). The protein molecule is a complex
structure made up of one or more chains of amino acid, which are linked by peptide bonds.
Proteins are essential constituents of the body; they form the structural material of
muscles, tissues, organs, etc. and are equally important as regulators of function, as
enzymes and hormones, proteins are synthesized in the body from their constituent amino
acids, which are obtained from the digestion of protein in the diet (Elizabeth and Martin,
2003).
CARBOHYDRATE: - One of a large group of compounds, including the sugar and
starch, that contain carbon, hydrogen and oxygen and have the general formular CX (H20)
Y- Carbohydrates are important as a source of energy: they are manufactured by plants
and obtained by animals from the diet, being one of the three main constituent of food. All
carbohydrates are eventually broken down in the body to the simple sugar glucose which
can
then take part in energy producing metabolic processes. Excess carbohydrate, not
immediately required by the body is stored in the liver and muscles in the form of
glycogen. In plants carbohydrate are important structural materials (e.g. cellulose and
storage products (commonly in the form of starch). (Elizabeth and Martin, 2003).
VITAMIN:- Any of a group of substances that are required in very small amounts, for
healthy growth and development: they cannot be synthesized by the body and are therefore
essential constituents of the diet. Vitamins are divided into two groups, according to
whether they are soluble in water or fat. The water soluble groups include the vitamin C;
the fat soluble vitamins are vitamins A, D, E and K. Lack of sufficient quantities of any of
the vitamins in the diet results in specific vitamin deficiency diseased (Elizabeth and
Martin, 2003).
FAT:- A substance that contains one or more fatty acids (in the form of triglyceride) and
is the principal form in which energy is stored by the body (in adipose tissue). It also
serves as an insulating material beneath the skin (in the subcutaneous tissue) and around
certain organs (including the
kidney). Fat is one of the three main constituents of food; it is necessary in the diet to
provide an adequate supply of essential fatty acid and from the efficient absorption of fat –
soluble vitamins from the intestine. Excessive deposition of fat in the body leads to
obesity. (Elizabeth and Martin, 2003).
A vendor is a person selling something (en.Wikipedia org/Wiki/vendor). The world Health
Organization (WHO) Indicated that food-borne diseases most of which are of microbial
origin are perhaps the most widespread problems in the contemporary world and this is
responsible for about one third of death world wide, through infectious conditions with
adverse effects can reduce economic productivity. Poor sanitary condition in most of the
local markets and the environment being highly polluted and charged with spoilage and
pathogenic flora is likely the source of contamination of food items sold by such vendors.
(Oweghe et al., 2001). It is known that poor hygienic conditions in a food environment
may encourage the multiplication of pathogenic organisms in food (Egeonu, 2002). It has
been observed that Bacillus cereus and Staphylococcus aureus grow to oxygenic levels in
food at 300c (Egeonu, 2003). Therefore microbiological examination of foods and food
contact surfaces may provide
information concerning the quality of the raw food, and the sanitary conditions under
which the food is processed (Michael et al; 2004). Microorganisms live throughout the
kitchen and can easily move around by attaching themselves to people easily move around
by attaching themselves to people, food and equipment. Bacteria may pass from
equipment to food which has not been properly cleaned and sanitized before being used to
prepare another food. Examination of food consumed and also wholesomeness. This
implies that the food to be consumed by humans should be pure and free from
contamination especially by pathogenic and spoilage micro organisms. Failure to ensure
the safety and wholesomeness of the food consumed by the public might lead to some
illness. To reduce contamination by microorganisms to a minimum level, and obtain good
keeping quality of the products, the raw materials should regularly be monitored and
examined.
Food contacts surfaces are a major concern for food service facilities in controlling the
spread of food-borne pathogens, surfaces such as bench tops, table, etc. may have bacterial
on them from contact with people, raw foods, dirty equipments or other things such as
cartons that have been stored
on the floor. If the bench tops are not properly cleaned, any food on them will be
contaminated by the bacterial (Kamil, 2005).
1.1 AIM
The overall aim of this work is to access selected foods sold by vendors in Ogbete main
market Enugu for bacterial contamination.
OBJECTIVES
1. To isolate and identify bacterial species associated with food contamination.

2. To determine the microbial load of isolated bacteria.

3. To establish the public health implication of consumption of such foods.

CHAPTER TWO
2.1 LITERATURE REVIEW
Food borne illness caused by microbial contamination of foods in an important
international public health problem and is known to be a major cause of diarrhea diseased
especially in developing countries (Mensah, 1997). In these developing countries a major
source of ready - to - eat foods are prepare and or sold at public places such as markets
place, schools, canteens and along the streets, all together termed street foods (SFS). The
SFS offer food at relatively cheaper cost and at easily accessible places. Furthermore, it
offers the traditional meals and preparations of a number of them are quite laborious and
time consuming. (Amoah, 1992; chakra Varky and Canet, 2002).
However, a number of observational studies have shown that these foods are sometimes
held at improper temperature, excessively handle by food vendors and sold at very dirty
surrounding (WHO, 2001, 2003; Ghosh et al., 2007). In addition the vendors practice poor
personal hygiene and
reports of food vendor being carriers and therefore could serve as a potential source of
transmission of enteric fevers are many. Most of the vendors have had either no formal
education or few years of schooling and therefore, lack knowledge on proper food
handling and their role in the transmission of pathogens (Mensah et al.,1999). At the same
time, most of the people who patronize these foods are more interested in its convenience
than question of its bacteriological quality and hygiene. The bacteriological quality of food
indicates the amount of bacterial contaminants it has; a high level of contamination
indicates low quality and more likely to transmit infection. The concerns have been raised
by the food and Agricultural organization (FAO) and other about these foods serving as a
potential source of food poisoning outbreaks (Chakravarty and Canet, 2002).
In Nigeria, consumption of street food has witnessed a phenomenal growth over the years
as rapid population growth over the years as rapid population growth, Urbanization,
Unemployment and poverty; occupational pressures and lifestyles changes has created a
poll of mobile and transient population who depend almost entirely on these relatively low
cost foods for their nutrition. (Martin, 2006).
Although epidemiological data on the incidence of forborne diseases are inadequate, and
the outbreak often not investigated, the recurrent episodes of food borne illnesses with
symptoms of gastro intestinal distress like diarrhea, vomiting, abdominal cramp and
nausea has remained a major cause of mortality and morbidity in Nigeria. (Nweze, 2010).
Chemicals heavily metals, parasites, fungi, viruses and bacteria can cause food – borne
illness, bacteria related food poisoning is the most common, but fewer than 20 of the
culprits. More than 90% of the cases of food poisoning each year are caused by
Staphylococcus aureus,
.Salmonella, Clostridium perfringes, Clostridium botulinum, Campylobacter, vibro
parahaemolyticus, Bacillus cerus and Entropathogenic Escherichia coli. These bacteria are
commonly found on many raw foods. Normally a large number of food – poisoning
bacteria must be presented to cause illness, therefore illness can be prevented controlling
number of bacteria present ,by preventing the small number from growing, destroying the
bacteria by proper cooking and avoiding re contamination (De Boer and Beuner,2011).
Poor personal hygiene, improper cleaning of storage and preparation areas and unclean
utensil course contamination of raw and cooked foods. Mishandling of raw and cooked
foods allows bacteria to grow. The temperature range in which most bacteria grow is
between 40 degrees (50c) and 1400f (600c). Raw and cooked foods should not be kept in
this danger zone any longer than absolutely necessary.
Analyzing foods for the presence of both pathogenic and spoilage bacteria is a standard
way of enduring food safety and quality (De Boer and Beuner, 2011). If micro organisms
are able to survive and grow on food which are sold and consumed by people, then the risk
of food borne – illness is increased in the society. The presence of micro organisms on
food can be important, because the essential nutrients of the food are ingested by some
organisms stimulate growth, while some organisms are known to be pathogenic to man as
long as their growth conditions are favorable (De Boer Beuner, 2011).
Bacteria may pass from equipment to food when the equipment that has touched the food
has not been properly cleaned and sanitized before being
used tom prepare another food (James, 2005). Food eaten has direct influence on health; it
is manufactures and food handlers to keep food safe from pathogenic micro organisms,
especially when such foods are to be consumed without further processing (Munide and
Kuria, 2005) when a food with harmful bacteria is ingested there is a period of time before
symptoms of the food – borne illness begin. The amount of times varies with the different
bacteria, how many consumed and the individual’s physical condition. Many different
harmful organisms produce the same symptoms. (FDA, 2004).
2.2 FOOD HYGIENE
Food hygiene is defined as a sanitary science which aims at producing food which is safe
for human consumption and of good keeping quality and this includes any sanitation
measures designed to prevent bacteria and other micro organisms of human origin from
reaching food stuff (Umoh and Odibo, 1999). Food hygiene is a subject of wide scope, it
aims at studying methods for production and preparation of food, which is safe and of
good quality. It covers not only the proper handling of every variety of food stuff
and drinks, but also food contact surfaces such as utensils, and apparatus used in the
preparation, services and consumption of the food and also the care to prevent
contamination with food poisoning bacteria which may originate from the animal or part
plant host supplying the food (Umoh and Odibo, 1999).
2.3 FACTORS THAT CONTRIBUTE TO FOOD –BORNE ILLNESS
They are, improper cooling of foods, time between preparing and serving, poor personal
hygiene, not cooking food properly, Abuse of the time temperature relationship, cross
contaminating raw and cooked foods.
Poor Personal Hygiene
Poor personal hygiene can result in food contamination for example when a food
personnel, fails to wash hands properly after using the restroom, toilet, is a serious risk of
faecal contamination (FDA, 2004). Everyone has bacteria on the skin, mouth, hands and so
many other organisms on various parts of the body like hair. Food service personnel can
contaminate food and cause food-borne illness. Food workers may transmit pathogens to
food from a contaminated surface, from one food to another
food or from hands contaminated with organisms from the gastrointestinal tracts (Munide
and Kuria, 2005). Therefore, hand contact with ready – to – eat food i.e. food that is edible
with out washing, cooking or additional preparation by the consumer pr by the food
establishment and that is expected to be consumed in that manner, represents a potentially
important mechanisms by which pathogens may enter the food supply. (Munide and Kuria,
2005).
Abuse Of The Time –Temperature Relationship
Abuse of time temperature relationship is also another factor that can cause food-borne
illnesses. To prevent food-borne illness, it is important to control the time that food is in
the temperature danger zone. This means hot foods should be kept at 1400F or above and
cold foods at 410For below (FDA, 2004). Don’t let cooked or refrigerated foods, such as
salads, sat at room temperature for more than two hours (FDA, 2004). Time temperature
relationship problems occur because
1. Food is not stored, prepared or held at the required temperature;
food is not cooked or reheated to temperature high enough to kill harmful micro organisms
2. Food is prepared in advance of service and proper temperature control is not maintained
(FDA, 2004).
Cross-Contaminating Raw And Cooked Food
Cross-contaminating raw and cooked food is transferring of harmful micro organisms from
a surface to food or from one food to another food. cross contamination can occur when
food contact surfaces is not cleaned or sanitized as necessary for food safety (FDA, 2004).
To prevent cross – contamination, it is important to wash hands with soap and warm water
before you start preparing food, before you handle a different food (for example, if you
just handled raw chicken, wash hands before preparing a salad), and after using the
bathroom. Don’t sneeze or cough on food. Organisms can “travel” from raw to cooked
food, so never let raw food touch cooked food. (FAD, 2004).
Persons At High Risk For Food-Borne Disease Are:
Infants and very young children
The elderly
Pregnant women
Individuals with weakened immune system like HIV, AIDS, liver disease or cancer. (FDA,
2004).
2.4 FEATURES OF COMMON FOOD –BORNE BACTERIA PATHOGENS

Salmonella species
Salmonella: - is a generic name applied to a group of nearly 2,000 biochemical related
serotypes responsible for food –borne illness. The disease is grossly underreported because
it is generally self- limiting gastroenteritis which may be misdiagnosed as intestinal
influenza by patient or the physician. As a consequence, estimates of the true incidence of
disease are based as assumptions derived from epidemiological evidence. Clearly,
salmonellosis continues to be an important cause of food-borne disease worldwide.
Two clinical manifestations caused by Salmonella are recognized:
enteric fever (a severe, life threatening illness) and the more common food-borne illness
syndrome: In both cased, the oral route.
Enteric fever, commonly referred to a typhoid fever, is primarily
caused by one species, Salmonella typhi. But other Salmonellae such as Salmonella
paratyphi are potentially capable of producing this syndrome. The illness is commonly
associated with foreign travel and affects an estimated 800 people annually (Mead et al;
1999). Although the route of entry of the pathogen into the body is primarily oral, the
symptoms of enteric fever are generally not elicited through the intestinal tract. However,
a short episode of vomiting and diarrhea sometimes occurs in the first day or two in
typhoid fever. The onset times vary considerably between typhoid and paratyphoid enteric
fevers. Onset time for typhoid is usually 8 – 15 days, seldom as short as five days but
sometimes as long as 30 – 35 days; while onset time for paratyphoid fever tends to be
shorter, and may be so short as to suggest typical food poisoning. (Parker, 1984).
Salmonella are destroyed at cooking contamination of cooked foods occurs from contact
with utensils that were not properly washed after use with raw products. If salmonella is
presented in raw or cooked foods, its growth can be controlled by
refrigeration below 400F. There are various environmental sources that include water, soil,
kitchen surfaces and animal faeces that helps in the transmission, Salmonella are
transmitted through the faecal matter of people or animals and are usually transmitted to
humans by eating foods that have been contaminated with faecal matter via cross-
contaminations. As few as 15 to 20 cells depending on the age and health of the host and
strain of bacterial are necessary to cause illness (FDA, 2004). It is estimated that
approximately 40,000 cases of Salmonelosis are reported each year in the U.S.A. (FDA,
2004).
Staphylococcus aureus
Man’s respiratory passage, skins and superficial wounds are
common sources of staphylococcus aureus. When Staphylococcus aureus is allowed to
grow in foods, it can produce a toxin that causes illness. Although cooking destroys the
bacteria, the toxin produce is heat stable and may not be destroyed. Staphylococcal food
poisoning occurs most often in foods that require hard preparation. Sometimes these types
of foods are left at room temperature for periods of time, allowing the bacteria to grow and
produce toxin. Good personal hygiene when handling foods will keep
Staphylococcus aureus out of foods and refrigeration of raw and cooked foods will prevent
the growth of these bacteria if any is present (Wagner, 2001)
Shigella
Shigellosis, or bacillary dysentery, as it is commonly known, is caused by bacteria of the
genus shigella¸ which include Shigella dysenteriae, shigella flexneri, shigella boydii and
shigella. Sonnei (Bryan, 1979). The normal habitat for shigella is the intestinal tract of
humans and other primates. Primarily mode of transmission appears to be person to-person
by the fecal-oral route (Feldman and Riley, 1985). Shigella is mostly associated with
chicken, raw vegetables, dairy products and poultry. Contamination of these foods is
usually through the feacal-Oral route and is most commonly due to faecally contaminated
water and unsanitary handling by food handlers (Todar, 2006). As few as 10 cells,
depending on the age and body condition of the host are necessary to cause disease. As
with Escherichia. coli, Shigella, are present in the diarrhea stool of infected person and can
be transmitted during infection as well as one to two weeks after symptom subsides, most
infections that occur are the
result of the bacterium passing from stools or of soiled fingers of one person to the mouth
or finger of another person. Shigella dysenteriae type cause deadly epidemics in
developing countries (CDC – DBM D, 2004). Shigella are transmitted through the faecal
matter of people or animals and are usually transmitted to humans by eating foods that
have
been contaminated with faecal matter through cross contamination. As few as 15 – 20
cells, depending on the age and health of the host and strain of bacteria are necessary to
cause food-borne illness. Generally, food-borne shigellosis is characterized by a high
attack rate, common-source epidemiology, and short incubation periods of 12 – 50 hours
(FDA/CFSA N, 2003b).
Enteropathogenic – Escherichia coli
A lactose – fermenting species is usually not harmful but some strains cause
gastrointestinal infections. Ingestion of the pathogenic serotype E. coli 0157 derived from
infected meat. Causes colitis with bloody diarrhea, which may give rise to the
complications of hemolytic uraemic syndrome (Elizabeth and Martin, 2003). E. coli is a
significant cause of diarrhea in developing
countries and localities of poor sanitation. Indeed it has been associated with “traveler
diarrhea”. However, the latest out break in North America occurred in a nursing home in
Ontario. There are at least four sub-group of enteropathogenic. Escherichia coli,
Enterolnvasive, Haemorrhagie and Enteropathogenic. Each strain has different
characteristics, the major source of the bacteria in the environment is probably the faces of
infected human but there may also be animal reservoirs and untreated water are the most
likely sources for contamination of food. E. coli 0157: H7 and its link to food become well
known to the public as a result of the 1993 E. coli 0157: H 7 outbreak caused by
contaminated hamburgers. Over 700 people become ill from this outbreak and four
children died (Buzby, 2001). E. coli 0157: H7 maybe acquired through consumption of
meat that has not been sufficiently cooked, and person – to person transmission can occur
via the faecal oral route E. coli 0157: H7 can be found in the diarrhea stool of infected
persons. The pathogens can be spread if personal hygiene and hand washing procedures
are inadequate. (Buzby, 2001).
SOME FOOD- AFFLIATED DISEASE SYMPTOMS
BORNE FOOD
BACTERIA AND
AFFILIATED
FOODS TABLE:1
MICROBE
1 Bacillus Meats, milk, B. cereus Diarrhea,
Cereus Rice, potato, Food Abdominal
And cheese poisoning cramps,
products Nausea.
2 Campylobacter Raw Chicken Campylobacter Diarrhea,
Jeini Unpasteurized iosis abdominal
Milk, Non- cramps, nausea
Chlorinated and fever,
Water Headache and
muscle pain
3 Clostridium Canned foods Food-borne Weakness,
botilinum including Botulism double vision,
vegetables and vertigo,
meats and difficulty in
soups. speaking,
swallowing
and breathing,
constipation.
4 Clostridium Non- Perfringens Severe
refrigerated food poisoning Abdominal
prepared foods
meats