BACTERIAL CONTAMINATION IN COOKED OR PROCESSED

FOOD

BY

BASHIR ABUBAKAR

20/03/01/176

FINAL YEAR RESEARCH PROJECT SUBMITTED TO THE

DEPARTMENT OF BIOTECHNOLOGICAL SCIENCES, FACULTY

OF SCIENCE, BORNO STATE UNIVERSITY IN PARTIAL

FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

DEGREE OF BACHELOR OF SCIENCE (B.Sc. HONS) IN

BIOTECHNOLOGY.

DECEMBER, 2023
 DECLARATION

I, BASHIR ABUBAKAR, hereby declare that the project work entitled BACTERIAL
CONTAMINATION IN COOKED OR PROCESSED FOOD is a record of an original work
done by me, as a result of my research effort carried out in the Faculty of Science, Borno State
University, under the supervision of Mr. H. M. CHIWAR

Student’s Signature & Date ________________________________

2
 CERTIFICATION

This is to certify that this study was carried out by Bashir Abubakar Matric Number
20/03/01/176 in the Department of Biotechnology, Faculty of Science, Borno State
University under my supervision.

_____________________________ ___________________________

PROJECT SUPERVISOR SIGN & DATE

_____________________________ ___________________________

HEAD OF DEPARTMENT SIGN & DATE

_____________________________ ___________________________

EXTERNAL EXAMINER SIGN & DATE

3
 DEDICATION

All praise is due to Almighty Allah (SWA) the master of creation and diverse who gives this
Wonderful opportunity to make it possible in due moment.

4
 ACKNOWLEDGEMENT

Thanks be to Almighty Allah (SWA) for his infinity mercy and protection right from the
beginning to the end of this project.

My profound gratitude goes to my project supervisor H M Chiwar, who supervised my project.

And also want to thank my Head of Department in person of Mrs. Fatimah M Maina as well as
entire staff of the Department.

Finally, I will like to thank my family, well wishers and friends for their prayer and support.

5
 Abstract
Bacterial contamination in cooked or processed foods is a major concern worldwide. It poses
serious health risks and can lead to foodborne illnesses, such as gastrointestinal infections.
Understanding the sources, types, and prevention methods of bacterial contamination is
crucial for safeguarding public health. One of the most common sources of bacterial
contamination is improper food handling and storage practices. The objectives of this study is
to check the presence and types of coliform, to identify presence of vibro cholerae as well as
determine the presence of Escherichia Coli. Methodology used includes, inclusion and
exclusion criteria, samples collections and preparation, sample innoculation, culturing as well
as Biochemical tests which also includes, Indole test, urease test and citrate test. At the end of
this research, the result from selected restaurants gives significant growth on, MacConkey
agar coliform is identified, while on thiosulfate Citrate Bile Salts Sucrose Agar (TCBS Agar)
determined Vibrio spp. respectively. Findings from this study show that, considerably some of
the commonly consumed cooked food in this study area, are contaminated with a wide
variety of bacteria, which may poses serious health risks and can lead to foodborne illnesses,
such as gastrointestinal infections, fever, diarrhea, nausea, abdominal pain, vomiting and
even more serious complications.

6
 TABLE CONTENT

Pages Title Page……………………………………………………………………………...........i

Declaration ……………………………………………………………………………………….ii

Certification ………………………………………………………………………………….…..iii
Dedication…………………………………………………………………………….………......iv
Acknowledgements…………………………………………………….……………………….....v

Abstract………………………………………………………………………….…………….....vi

Table of contents ……………………………………………………………………………......vii

CHAPTER ONE

1.0 INTRODUCTION ………………………………………………………………………1

1.1 BACKGROUND OF THE STUDY………………………………………………….1

1.2 STATEMENT OF PROBLEMS ……………………………………………………..2

1.4 AIMS AND OBJECTIVES OF RESEARCH ……………………………………..2

CHAPTER TWO

2.0. LITERATURE REVIEW ……………………………………………………………...4

2.1 BACTERIAL CONTAMINATION…………………………………………………..4

2.2 Scientific Classification of Escherichia coli………………………………………....5

2.2.1 Taxonomy of Escherichia coli……………………………………………………….5

7
2.2.2 Characteristics of Escherichia coli………………………………………………….6

2.2.3 EPIDEMIOLOGY of Escherichia coli…………………………………………….6

2.3 Scientific Classification of Klebsiella ……………………………………………….7

2.3.1 Characteristics of Klebsiella…………………………………………………………8

2.3.2 Taxonomy of Klebsiella………………………………………………………………8

2.3.3 Epidemiology of Klebsiella………………………………………………………….9

2.4 Scientific Classification of Salmonella……………………………………………....9

2.4.1 Characteristics of Salmonella………………………………………………………10

2.4.2 Taxonomy of Salmonella……………………………………………………………10

2.4.3 Epidemiology of Salmonella……………………………………………………….10

2.5 Scientific Classification of Shigella………………………………………………….11

2.5.1 Characteristics of Shigella ………………………………………………………….11

2.5.2 Epidemiology of Shigella …………………………………………………………..12

2.6 GROWTH OF FOODS BORNE BACTERIA…………………………………….12

2.6.1 GROWTH OF FOODS BORNE BACTERIA …………………………….13

2.6.2 FOOD PREFERRED BY FOOD BORNE BACTERIA……………………...13

2.6.3 PEOPLE AT RISK OF FOOD BORNE ILLNESS……………………………14

2.6.4 OCCURENCE OF FOOD BORNE ILLNESS……………………………….14

2.6.5 CLINICAL SYMPTOMS OF FOOD BORNE ILLNESS……………………15

8
2.6.6 COMMON PREVENTIVE MEASURES OF FOOD BORNE ILLNESS…15

2.7 FOOD BORNE PATHOGENS…………………………………………………………….15

2.7.1 Escherichia coli…………………………………………………………………….16

2.7.2 Klebsiella………………………………………………………………………………16

2.7.3 Salmonella………………………………………………………………………….….16

2.7.4 Shigella ………………………………………………………………………….…..17

2.7.5 Clostridium perfringens……………………………………………………………..18

2.7.6 Campylobacter………………………………………………………………………..18

2.7.7 Listeria monocytogenes……………………………………………………………..19

2.8 FOOD BORNE TOXINS AND OTHER CHEMICALS ……………………….19

2.8.1 Plant toxins…………………………………………………………………………….19

2.8.2 TOXIC CHEMICALS AND OTHER METALS……………………………….20

CHAPTER THREE

3.0 METHODOLOGY……………………………………………………………………..21

3.1 STUDY AREA …………………………………………………………………………21

3.2 INCLUSION AND EXCLUSION CRITERIA …………………………………..21

3.3 SAMPLES COLLECTION AND PREPARATION…………………………….21

3.4 CULTURING METHOD OR SAMPLE INNOCULATION………………….22

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3.6 Biochemical Tests……………………………………………………………………..22

3.6.1 INDOLE TEST……………………………………………………………………..22

3.6.2 UREASE TEST……………………………………………………………………..22

3.6.3 CITRATE UTILIZATION TEST ……………………………………………….23

CHAPTER FOUR

4.0 RESULT…………………………………………………………………………….24

Table: 4.1 INITIAL CULTURE OF MACCONKEY AGAR……………………..24

4.2 BIOCHEMICAL TESTS ……………………………………………………………25
4.3 INITIAL CULTURE ON TCBS. …………………………………………………………25
Table: 4.4 SUBCULTURE ON TCBS. ………………………………………………………26
CHAPTER FIVE
5.1 DISCUSSION………………………………………………………………………….27
5.2 CONCLUSION ………………………………………………………………………..28

5.3 RECOMMENDATIONS …………………………………………………………28

REFERENCES………………………………………………………………………………30

APPENDIX………………………………………………………………………….………….41

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 CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Bacterial contamination in cooked or processed foods is a major concern worldwide. It poses

serious health risks and can lead to foodborne illnesses, such as gastrointestinal infections.
Understanding the sources, types, and prevention methods of bacterial contamination is crucial
for safeguarding public health. One of the most common sources of bacterial contamination is
improper food handling and storage practices (Jain et al., 2020). There are different types of
contaminations that can cause cases of food poisoning and it is usually difficult to detect the
sources. Often, food smells and taste normal but May actually contain bacteria, chemicals, or
viruses (Marl, 2015).

When cooked or processed foods are not stored at the correct temperatures, bacteria can multiply
rapidly, leading to the growth of harmful pathogens. Some of the most common bacteria
associated with foodborne illnesses include Salmonella, Escherichia coli (E. coli) as well as
Campylobacter resulting in fever, diarrhea, nausea, abdominal pain, vomiting and even more
serious complications. (Majowicz et. al., 2014). Therefore according to the study conducted by
the Centers for Disease Control and Prevention (CDC, 2019), bacterial contamination was
identified as the cause of approximately 48 million cases of foodborne illnesses in the United
States alone (CDC, 2019). This alarming statistic highlights the importance of addressing this
issue effectively. To prevent bacterial contamination in cooked or processed foods, several
measures can be taken.

Firstly, ensuring proper food hygiene practices, such as frequent hand washing, the use of clean
utensils, and thorough cooking of food, is essential. Additionally, implementing and adhering to
the Hazard Analysis Critical Control Point (HACCP) system, a preventive approach to food
safety can significantly reduce the risk of bacterial contamination (FAO, 2018).

Furthermore, adequate storage and temperature control are vital. Refrigeration of perishable
foods below 5 degrees Celsius (41 degrees Fahrenheit) and freezing at or below -18 degrees

11
Celsius (0 degrees Fahrenheit) can inhibit bacterial growth (FDA, 2017). It is also crucial to
prevent cross-contamination by separating raw and cooked foods, using different cutting boards,
and properly sanitizing all surfaces and equipment. The burden of disease can be defined as the
incidence and prevalence of morbidity, disability, and mortality associated with acute and
chronic manifestations of diseases (WHO, 2006). More than 200 types of diseases (cholera,
salmonellosis, shigellosis, typhoid, and other gastroenteritis diseases) are estimated to be caused
or spread by food, occasionally causing long-term health problems in vulnerable groups such as
the elderly, pregnant women, children, and immune compromised people (Loukieh et al., 2018;
WHO, 2019).

1.2 STATEMENT OF PROBLEMS

Due to the significance of controlling the outbreak of pathogenic microorganism, food contact
surfaces in food production and catering establishments are the main concern. Current statistics
of food borne illness in various industrialized countries shows that up to 60% of cases may be
caused by poor food handling and by contaminated foods served in food service establishments.

In 1989, it was estimated that the total cost of bacterial food borne illness to the United States

economy was over $6Billion. Hence it is a burden on the economy. In developing countries like

Nigeria, the effect on economic activities and development can only be far more severe (Ali et al,

2017).

Considering the importance of these issues, the present research aimed to assess the

bacteriological analysis of food contact surfaces from some selected restaurants in Maiduguri

metropolitan area of Borno state,Nigeria with the zeal to contribute towards providing a solution

towards these health problems.

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1.4 AIMS AND OBJECTIVES OF RESEARCH

Aims

This research aimed at assessing bacterial contamination in cooked or processed foods from
selected restaurants in Maiduguri, Borno state.

Objectives

The specific objective of this research is to;

1. To check the presence of coliform Bacteria

2. To identify the type of coliform

3. To identify the presence of vibro cholerae

4. To determine the presence of E.coli

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 CHAPTER TWO

2.0. LITERATURE REVIEW

2.1 BACTERIAL CONTAMINATION

There are different types of contaminations that can cause cases of food poisoning and it is
usually difficult to detect the sources. Often, food smells and taste normal but May actually
contain bacteria, chemicals or viruses (Marl, 2015).

Bacterial contamination in cooked or processed foods is a major concern worldwide. It poses

serious health risks and can lead to foodborne illnesses, such as gastrointestinal infections.
Understanding the sources, types, and prevention methods of bacterial contamination is crucial
for safeguarding public health. One of the most common sources of bacterial contamination is
improper food handling and storage practices (Jain et al., 2020).

When cooked or processed foods are not stored at the correct temperatures, bacteria can multiply
rapidly, leading to the growth of harmful pathogens. Some of the most common bacteria
associated with foodborne illnesses include Salmonella, Escherichia coli (E. coli) as well as
Campylobacter resulting in fever, diarrhea, nausea, abdominal pain, vomiting and even more
serious complications. (Majowicz, et. al, 2014)

Food borne diseases is a growing public health problem worldwide and has brought considerable
economic burdens to hospitals and other healthcare costs (Chen et al., 2019). According to the
reports of the United States Centers for Disease Control and Prevention (US-CDC), the incidence
of foodborne diseases in the United States is one in six cases, which results in 128,000
hospitalizations and 3,000 deaths each year.

Cooked or processed foods refers ready-to eat (RTE) foods, prepared food which can be
consumed immediately or after taking a few steps such as heating before consuming
(Microbiological Guidelines, 2007 &Thienhirun chun 2018).

14
2.2 Scientific Classification of Escherichia coli

Domain Bacteria

Phylum Proteobacteria

Class Gamma proteobacteria

Order Enterobacteriales

Family Enterobacteriaceae

Genus Escherichia

Species coli

Escherichia coli are just one of the many bacteria that cause diarrhea. Escherichia coli was
originally called Bacterium coli commune which was first isolated in 1885 by Theodor Escherich
from the faeces of a child but it was not until 1945 that Bray and other researchers demonstrated
its involvement in the gastroenteritis (Marianne, 2003).

Escherichia coli are common inhabitant of the Gastrointestinal Tract (GIT) of humans and
animals. There are strains of Escherichia coli that are harmless commensals of the intestinal tract
and others that are major pathogens of human and animals. The pathogenic Escherichia coli can
be divided into those strains causing diseases inside the intestinal tract and others capable of
infection at extra-intestinal sites (Rodney, 2006).

2.2.1 Taxonomy of Escherichia coli

Escherichia coli include a vast population of bacteria and they demonstrate a high degree of
phenotypic and genetic diversity. In 1895, Migula reclassified the bacteria belonging to the
genus Escherichia which was named so after discovery. The genus Escherichia belongs to the
bacterial group called coliforms which are members of the enteric and are known as the
Enterobacteriaceae (Islam et al., 2014).

15
 Besides Escherichia coli, there are other species belonging to the genus Escherichia and
they include E. blattae, E. hermanii, E. adecarboxylata, E. fergusonii, and E. vulneris. The
evolution of independent pathogenic type of Escherichia coli is striking and to date unmatched
by any other bacterial genus (Rodney, 2006).

2.2.2 Characteristics of Escherichia coli

Escherichia coli are the ubiquitous bacterial species and commensal of human and warm-
blooded animals. They are Gram negative, facultative anaerobes, non-spore forming, and straight
rods. They are arranged in pairs and some strains are motile with peritrichous flagella while
others are non-motile having capsules or microcapsules. They are catalase positive, oxidase
negative, they can reduce nitrate. Approximately 95% of Escherichia coli strains are indole and
methyl red positive, but they are Voges-Proskauer and citrate negative.

They are harmless commensals of the human digestive tract and can also be found in other
warm-blooded animals but they can be versatile pathogens in immune compromised patients.
Escherichia coli have been used as an indicator of fecal contamination in food and water due to
its common occurrence in faeces and its survival in water (Bachir and Abouni, 2015).

Most Escherichia coli strains are capable of growing over a wide range of temperature of
about 15-48°C but the optimum growth temperature is 37°C. They can also grow within a pH
range of about 5.5-8.0 but best grow at neutrality (Rodney, 2006).

Based on evolutionary relatedness, Escherichia coli are subdivided into serotypes and
these serotypes are based on the antigen present in its surface. The antigen are somatic antigen
(O-antigen which is part of the lipopolysaccharide layer and k-antigen) and flagella antigen
which is the H-antigen (Wei et al., 2014). Escherichia coli have over 150 antigenically unique
O-antigens, four forms of K-antigens based on physical, biochemical and genetic criteria, and 53
H-antigen.

2.2.3 EPIDEMIOLOGY of Escherichia coli

Although regarded as part of the flora of the human intestinal tract, several highly adapted
Escherichia coli clones have evolved and developed the ability to cause disease in several areas
16
of the human body. Most of these diseases are related to mucosal surfaces. Sometimes however,
mucosal colonization of the intestine and urinary tract may be asymptomatic.

In developing countries, contaminated water is a principal vehicle for transmission of

Escherichia coli infection either by ingestion or through contaminated foods that have been
irrigated, washed, or prepared with contaminated water. This is less common in developed
countries with higher standards of general hygiene. However, drinking water contaminated by
sewage or animal waste has been implicated in outbreaks, and in one case, water in a paddling
pool was suspected as the means of spreading infection to children. As food and person-to-
person contact are alternative sources of exposure, the role of the water may be difficult to prove.
The most important determinant of risk is the destinations of the traveler. Attack rates of 20%-
50% are commonly reported. High-risk destination includes most of the developing countries of
Central America, Africa, Middle East and Asia. Intermediate-risk destinations include most of
the Southern European countries and a few Caribbean islands. Low-risk destinations include
Canada, Northern Europe, Australia, New Zealand, the United States and a number of Caribbean
island (Marianne, 2003).

There are five different classes of Escherichia coli and they cause disease in different host. The
classes include: Enterotoxigenic E. coli (ETEC) which causes disease in humans, pigs, sheep and
cattle; Enteropathogenic E. coli (EPEC) which causes disease in human, rabbit, dogs etc.
Enterohemorrhagic E. coli (EHEC) which cause disease in human, cattle and goat; Enteroinvase
E. coli (EIEC) found only in human; Enteroaggregative E. coli (EAEC) also found in humans
only (Bachir and Abouni, 2015).

2.3 Scientific Classification of Klebsiella

Kingdom: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Enterobacterales

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Family: Enterobacteriaceae

Genus: Klebsiella

Friedlander described Klebsiella bacillus which is why it was termed Friedlander bacillus for
many years. The members of the genus Klebsiella are a part of the human and animal's normal
flora in the nose, mouth and intestines. The species of Klebsiella are all gram-negative and
usually non-motile. They tend to be shorter and thicker when compared to others in the family
Enterobacteriaceae. The cells are rods in shape and generally measures 0.3 to 1.5 µm wide by 0.5
to 5.0 µm long. They can be found singly, in pairs, in chains or linked end to end. Klebsiella can
grow on ordinary lab medium and do not have special growth requirements, like the other
members of Enterobacteriaceae. The species are aerobic but facultatively anaerobic. Their ideal
growth temperature is 35° to 37 °C, while their ideal pH level is about 7.2.( Ristucci, et al
1984 ).Klebsiella is classified under the Enterobacteriaceae family which contained a large array
of biochemically distinct genus, including the model organism Escherichia coli and the notorious
human pathogens Salmonella, Yersinia, Serratia, Enterobacter, Citrobacter, Kluyvera, Leclercia,
Raoultella, Cronobacter, etc.

2.3.1 Characteristics of Klebsiella

Klebsiella is a Gram-negative, non-motile, facultative anaerobic bacterium. It is capable of

fermenting glucose and is often found in the human intestinal tract. Some species, such as
Klebsiella pneumoniae, are known to produce a capsule, which contributes to their virulence and
resistance to desiccation. (Smith, J. 2023).

2.3.2 Taxonomy of Klebsiella

The genus Klebsiella belongs to the tribe Klebsiellae, a member of the family
Enterobacteriaceae. The organisms are named after Edwin Klebs, a 19th century German
microbiologist. Klebsiellae are nonmotile, rod-shaped, gram-negative bacteria with a prominent
polysaccharide capsule.

2.3.3 Epidemiology of Klebsiella

18
Klebsiella species are found everywhere in nature. This is thought to be due to distinct
sublineages developing specific niche adaptations, with associated biochemical adaptations
which make them better suited to a particular environment. They can be found in water, soil,
plants, insects and other animals including humans (Bagley S 1985).

In healthcare settings, Klebsiella pneumoniae is known for causing nosocomial infections,

particularly in immunocompromised individuals, and is associated with healthcare-acquired
pneumonia, bloodstream infections, and urinary tract infections.

2.4 Scientific Classification of Salmonella:

Kingdom: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Enterobacterales

Family: Enterobacteriaceae

Genus: Salmonella

Species: Salmonella enterica, Salmonella bongori etc.

Salmonella was first discovered and isolated from the intestines of pigs infected with classical
swine fever, by Theobald Smith in 1855. The bacterial strain was named after Dr Daniel Elmer
Salmon, an American pathologist who worked with Smith. The nomenclature of Salmonella is
controversial and still evolving. Currently, the Centers for Disease Control and Prevention (CDC)
uses the nomenclatural system of Salmonella recommended by the World Health Organization
(WHO) Collaborating Centre (Popoff et al. Citation2003). According to this system, the genus
Salmonella is classified into two species, Salmonella enterica (type species) and Salmonella
bongori, based on differences in their 16S rRNA sequence analysis. The type species, S. enterica,
can be further classified into six subspecies based on their genomic relatedness and biochemical
properties (Reeves et al. Citation1989)

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2.4.1 characteristics Of Salmonella

Salmonellae are Gram-negative, flagellated, facultatively anaerobic bacilli possessing three

major antigens: H or flagellar antigen; O or somatic antigen; and Vi antigen (possessed by only a
few serovars). H antigen may occur in either or both of two forms, called phase 1 and phase 2.

2.4.2 Taxonomy of Salmonella

Salmonella was first observed in a patient who died of typhoid fever in 1880 and was known as
Typhoid bacillus. In 1884, it was isolated from the intestine of pigs and named Bacillus
choleraesuis. The name was later changed to Salmonella Choraesuis in honor of D.E. Salmon. In
1966, Kauffmann proposed a one serotype one specie concept for the genus Salmonella based on
the identification of O and H antigens, resulting in about 3000 species of Salmonella if applied
today. Multiple Salmonella species existed and were taxonomically accepted before 1973.

2.4.3 Epidemiology of Salmonella

In 2000, the incidence of enteric fever was estimated to be 22 million cases resulting in 200,000
deaths worldwide, predominantly in underdeveloped countries (Crump et al. Citation2004). The
incidence and mortality rate of enteric fever vary from region to region, but the mortality rate
can be as high as 7% in spite of antibiotic therapy.

Enteric fever is endemic in many regions of the African and Asian continents as well as
countries such as in Europe, South and Central America, and the Middle East. The incidence of
enteric fever in the USA and some European countries is low, with the total number of
Salmonella cases being less than 10 per 100,000 annually. Most of the cases reported in these
countries are related to travel, with the disease being imported by foreigners or travellers
returning from Africa, India or Pakistan (Molbak et al. Citation2002; Cooke et al. Citation2007).

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2.5 Scientific Classification of Shigella:

Kingdom: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Enterobacterales

Family: Enterobacteriaceae

Genus: Shigella

Species: Shigella dysenteriae, Shigella flexneri, etc.

Shigella is a genus of bacteria that belongs to the family Enterobacteriaceae, which also includes
other well-known bacteria such as Escherichia coli and Salmonella. Within the genus Shigella,
there are four recognized species: Shigella dysenteriae, Shigella flexneri, Shigella boydii, and
Shigella sonnei. These species are further divided into multiple subgroups and strains based on
specific characteristics, such as surface antigens and genetic differences. For example, the
species Shigella flexneri has several subtypes, designated by Roman numerals, such as Shigella
flexneri 1, Shigella flexneri 2, and so on. Each of these subtypes may have slightly different
properties and be associated with specific patterns of disease (Popoff et al. Citation2003).

2.5.1 Characteristics of Shigella

Shigella are classified as Gram-negative due to their cell wall composition. The bacteria are rod-
shaped (bacilli). They are Non-motile Unlike many other Gram-negative bacteria.

Shigella species do not have flagella and are therefore non-motile, Facultative anaerobes, they
can grow in the presence or absence of oxygen.

Shigella primarily affects humans and primates, causing an illness known as shigellosis or
bacillary dysentery (Crump et al. Citation2004).

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2.5.2 Epidemiology of Shigella

Shigella is a major cause of diarrheal disease worldwide, particularly in low- and middle-income
countries with poor sanitation and hygiene conditions. The bacteria are primarily transmitted
through the fecal-oral route, often via contaminated food and water. Factors such as
overcrowding, inadequate sanitation, and poor personal hygiene contribute to the rapid spread of
the disease in communities (CDC, 2020).

Children, especially those under the age of 5, are particularly susceptible to Shigella infection.
This vulnerability is due to their immature immune systems and higher likelihood of contact with
contaminated environments. Additionally, travelers to countries with poor sanitation may also be
at risk of acquiring shigellosis (CDC, 2020).

Epidemiological studies have highlighted the seasonal patterns of Shigella infections, with higher
incidence during the warmer months. This can be attributed to factors such as increased bacterial
proliferation in higher temperatures and changes in water and food availability.

Moreover, antimicrobial resistance is a growing concern in the epidemiology of Shigella. The

emergence of multidrug-resistant strains of Shigella poses a significant public health threat and
underscores the importance of prudent antibiotic use and surveillance of resistance patterns
(Crump et al. Citation2004).

Shigella is a group of bacteria that can cause gastroenteritis in humans. It is a significant cause of
diarrhea worldwide, especially in areas with poor sanitation and hygiene (Crump et al.
Citation2004).

2.6 GROWTH OF FOODS BORNE BACTERIA

When certain disease-causing bacteria or pathogens contaminate food, they cause food borne

illness often called food ‘‘poisoning’’. Food borne infection is caused as a result of the of the

actual pathogen or its product (toxins) and other chemicals in a food at a concentration

22
considerable enough to cause a disease. Foods that are contaminated may not look, taste or smell

any different from foods that are safe to eat.

Diarrheal diseases are linked to the death of an estimated 2 million people annually-mostly

children and most of this illness, including food borne illness, are attributed to contaminated food

or water. Salmonella, campylobacter, listeria and Escherichia coli are the most common bacteria

causing food borne illness. Unfortunately, some food borne bacteria such as bacillus cereus,

staphylococcus aureus produces toxins that are heat-resistance, which means they cannot be

destroyed by cooking. The virus that most commonly causes gastrointestinal illness is norovirus

which can be transmitted through contaminated food or water, as well as contaminated surfaces

such as sinks, tables, handrails etc. Food borne illness can be serious or fatal.

2.6.1 GROWTH OF FOODS BORNE BACTERIA

Food borne bacteria are often present in food and in the right conditions, a single bacterium can

grow into more than two million bacteria in just seven hours.

These bacteria multiply in foods with lots of carbohydrates and protein content when the

food temperature is between 5-60°c which is often known as food danger zone. Some food borne

bacteria can grow under refrigerator in ready-to-eat food and listeria monocytogenes is one of

such bacteria. Staphylococcal bacteria can grow in food where they produce toxins and as such,

staphylococcal food poisoning does not result from the ingestion of the pathogen but rather by

the ingestion of the toxins produce by the bacteria that are already present in the contaminated

food.

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2.6.2 FOOD PREFERRED BY FOOD BORNE BACTERIA

Bacteria can grow and multiply on some type of food more easily than others. The type of food

which are most preferred by food borne pathogens include those with high content of

carbohydrate and/proteins. These foods include:

1. Meat

2. Poultry and products

3. Dairy products

4. Seafood

5. Cooked rice

6. Prepared fruits

7. Potato salad.

The above foods are more likely to be infected by food borne bacteria but other foods can

also be infected or cross contaminated by them if appropriate food safety measures are not taken

during the course of the preparation, storage, transportation and especially, handling of ready-to-

eat foods.

2.6.3 PEOPLE AT RISK OF FOOD BORNE ILLNESS

Some people are at high risk for developing food borne illness. These categories of people

include;

1. Young children

2. Pregnant women

24
 3. Older adults

4. People with weakened immune system e.g. HIV patients.

2.6.4 OCCURENCE OF FOOD BORNE ILLNESS

Food borne illness usually follows within 1-3days after the consumption of contaminated food

during a party or festival. Its occurrence is often in a cluster where person serve themselves

rather than being served by a single server although, cases from single server do occur mostly as

a result of poor handling techniques and hygiene.

The following conditions may be responsible for food borne illness

1. Not cooking food thoroughly especially meat and meat products

2. Improper refrigeration temperature for foods that need to be chilled i.e. at below 5°c

3. Keeping cooked food unrefrigerated for more than more than an hour

4. Refrigerating food, holding and then, consumption of such foods

5. Eating food that has been touched by someone with diarrhea and vomiting

6. Cross-contamination such as placing food on plates that had raw meat.

2.6.5 CLINICAL SYMPTOMS OF FOOD BORNE ILLNESS

Food borne illness is presented with some combination of nausea, vomiting and diarrhea that

may or may not be bloody, sometimes with other symptoms. After eating tainted food,

abdominal cramps, diarrhea and vomiting can start as early as one hour or even 3days depending

on the food borne pathogen, the type of toxin and the level of food contaminated.

25
2.6.6 COMMON PREVENTIVE MEASURES OF FOOD BORNE ILLNESS

A few simple actions can cut the likelihood of food borne illness drastically. These can be

summarized into five key steps for safer food as stated by World Health Organization (WHO).

1. Keep clean; these include thorough washing of raw fruits and vegetables, hands and

cutting boards clean.

2. Separate raw and cooked food; raw foods and ready-to-eat food should not be mixed also,

raw fish, meat and raw vegetables should be also separated

3. Cook thoroughly; all meat, poultry and sea foods should be cooked thoroughly especially

shellfish; reheating of all leftovers until they are steaming hot.

4. Keep food at safe temperature; all prepared food should be refrigerated within two hours

of preparation, food should never be defrosted at room temperature, it should be done in

the refrigerator, cold water or microwaves.

5. Use of safe water and raw materials; use of safe water for the preparation of food and

also checking labels of packed foods while buying.

2.7 FOOD BORNE PATHOGENS

According to the Centre for disease control and prevention, approximately 48 million Americans

got sick, 128,000 are hospitalized and 3,000 die each year from food poisoning.

Bacteria, viruses and parasites are the sources of many food poisoning cases, usually due to

improper food handling, some bacteria, in small amounts, are not harmful to most healthy adults

because the human body is equipped to fight them off. The trouble begins when certain bacteria

and other harmful pathogens multiply and spread, which can happen when food is mishandled.

26
Foods that are contaminated may not look, taste or smell any different from foods that are safe to

eat. Symptoms of food poisoning vary and develop as quickly as 30 minutes to as long as several

days after eating food that has been infected.

As identified by the CDC, eight known pathogens (bacteria, viruses and parasites) account for

the majority of food borne illness, hospitalization and death in the world.

2.7.1 Escherichia coli

Escherichia coli better known as E. coli are a large group of bacteria. Although most strains of E.

coli are harmless, some can make one very sick. One strain, E. coli O157:H7 (STEC) is

commonly associated with food poisoning outbreaks because its effect can be extremely severe.

Sources: This includes eating raw meat or undercooked ground beef or taking unpasteurized

dairy products or beverages.

Prevention: Wash your hands, cook meat and poultry thoroughly, avoid unpasteurized dairy

products, keep cooking surfaces clean and prevent cross contamination. Also, do not swallow

water when playing in pools, lakes or streams.

2.7.2 Klebsiella

The primary sources of Klebsiella as foodborne pathogens are typically associated with

contamination of food products during food handling, storage, or processing. Here is some

information on the sources and preventive measures for Klebsiella as foodborne pathogens:

Sources: Raw Vegetables, Improperly Cooked Foods.

27
Preventive: Good Hygiene Practices, Proper Food Handling and Storage, Cooking and

Pasteurization.

2.7.3 Salmonella

Salmonella is the name of a group of bacteria that causes the infection known as salmonellosis. It

is one of the most common bacterial cause of diarrhea and the most common cause of food borne

related hospitalizations and deaths. Salmonella is more severe in pregnant women, older aduts,

younger children and those with weakened immune system. Salmonella bacteria can live in the

intestinal tract of humans and other animals, it can spread easily unless you use proper hygiene

and appropriate cooking methods.

Sources: One can contract salmonellosis by consuming raw and undercooked eggs,

undercooked poultry and meat, contaminated raw fruits and vegetables (such as sprouts and

melons), as well as unpasteurized milk and other dairy products. It can be transmitted through

contact with infected animals or infected food handlers who have not washed their hands after

using the bathroom.

Prevention: Cook foods such as eggs, poultry and ground beef thoroughly at recommended

temperatures. Wash raw fruits and vegetables before peeling, cutting or eating. Avoid

unpasteurized dairy products and raw or uncooked meats, poultry and sea foods. Wash hands

often, especially after handling raw meats or poultry. Clean kitchen surfaces and avoid cross

contamination.

28
2.7.4 Shigella

Shigella is a group of bacteria that can cause foodborne illness when ingested. Here's some

information on the sources and preventive measures for Shigella as foodborne pathogens:

Sources: Contaminated Water, Poor Food Handling Practices,

Preventive: Thorough Hand Washing, Safe Water Sources, . Proper Food Hygiene etc

2.7.5 Clostridium perfringens

Clostridium perfringens also known as C. perfringens is very common in our environment. It can

multiply very quickly under ideal conditions. Infants, young children and older adults are most at

risk.

Sources: Illness usually occurs by eating food contaminated with large number of this

bacterium that produces enough toxins to cause sickness in the form of abdominal cramping and

diarrhea. C perfringens is sometimes referred to as the ‘‘buffet germ’’ because it grows faster in

large portion of food, such as casseroles, stews and gravies that have been sitting at room

temperature in the danger zone. If food is not originally cooked, reheated or kept at the

appropriate temperature, live bacteria may be consumed and cause illness.

Prevention: Cook food thoroughly and keep it out of the danger zone, above a temperature of

60°c or below 4°c. Practice leftover safety by dividing roast and stews into smaller quantities

when refrigerating for faster cooling. Leftovers should be reheated to an internal temperature of

75°c or higher before serving.

29
2.7.6 Campylobacter

Campylobacter is a common cause of diarrhea. Most cases of campylobacteriosis, the infection

caused by campylobacter bacteria, are associated with eating raw or uncooked food, poultry and

meat or from cross contamination of other foods by these items. Freezing reduces the number of

campylobacter bacteria on raw foods but does not kill them completely, so proper heating of

foods is important. Campylobacteriosis occurs more frequently in the summer and is most

common in infants and young children.

Sources: Consuming raw and undercooked poultry and meats, unpasteurized dairy products

and untreated water or contaminated produce.

Prevention: Cook all foods thoroughly, prevent cross-contamination by using separate cutting

boards when handling raw and cooked foods, do not drink unpasteurized milk or untreated water

and wash hands frequently. Wash raw fruits and vegetables before peeling, cutting and eating.

2.7.7 Listeria monocytogenes

Eating food contaminated with Listeria monocytogenes causes listeriosis, a serious infection that

primarily affects individuals who are at high risk for food poisoning: older adults, pregnant

women, young children and people with weakened immune system. Listeria can grow at

refrigerator temperatures where most other bacteria cannot grow.

Sources: Listeria is found in refrigerated, ready to eat food such as hot dogs, deli meats,

unpasteurized milk, raw sprouts, dairy products and raw or undercooked meat, poultry and sea

food.

30
Prevention: Cook all foods to proper temperatures and reheat precooked food to 165⁰F (75⁰C);

wash raw fruits and vegetables before peeling, cutting or eating; separate uncooked meat and

poultry from foods that are already cooked or ready to eat; wash hands thoroughly; store food

safely; maintain a clean refrigerator and kitchen area; wash reusable grocery regularly.

2.8 FOOD BORNE TOXINS AND OTHER CHEMICALS

Seafood toxins are not destroyed by heat. Ciguatera fish poisoning is caused when we consume

fish that have fed on toxic algae. The symptoms are a combination of gastrointestinal,

neurological, and cardiovascular. This illness is usually self-limiting. Scombroid poisoning is

caused when consume fish contaminated with histamines. These histamines are produced by

bacteria on the fish. It is important to point out that this type of poisoning can also occur with

other foods that have appropriate amino acid and bacteria. The symptoms of this illness generally

begin with tingling or burning sensations in the mouth. Symptoms can be severe enough to

require hospitalization, especially in the elderly.

2.8.1 Plant toxins

Plant toxins are most destroyed by heat. Examples include certain species of mushrooms,

aflatoxins produced by fungi contaminated grains and hemlock and jimsonweed or toxic plants.

2.8.2 TOXIC CHEMICALS AND OTHER METALS

Toxic chemicals and metals have resulted in food borne illness. Zinc from using

galvanized containers has resulted in zinc poisoning. Copper may leach from copper plumbing

and cause people to become ill from copper poisoning. Antimony poisoning may result from the

31
use of enamel ware. Lead poisoning may result in illness from lead leaching from plumbing or

solder.

32
 CHAPTER THREE

3.0 METHODOLOGY

3.1 STUDY AREA

The study was carried out in Maiduguri metropolitan area of Borno state. Borno borders the

Republic of Niger to the north, Lake Chad (and the Republic of Chad) to the northeast, and

Cameroon to the east; on the south and west it borders the Nigerian states of Adamawa, Gombe,

and Yobe.

Borno State (Motto: Home of Peace), is a state in north-eastern Nigeria. Its capital is Maiduguri,

Borno state, Nigeria lays at latitude 11030N and longitude 13000E with a total land mass area of

57,799 square kilometer (km2) (22,316 square meter (sqm)) which makes it the 2nd of 36 states

in the country by area rank (Aborisade, 2001). From the2006 Census population figures, it is

ranked 12th of 36 states with density of 72/km2 (190/sq m) and a total population of 4,171,104

million people.

3.2 INCLUSION AND EXCLUSION CRITERIA

Fresh cooked foods from Borno State University, Borno Express, Tanker Park, Post Office,

Tashan Kano as well as University of Maiduguri teaching Hospital (UMTH) were included while

spoiled, and cooked foods remains were excluded from the study.

33
3.3 SAMPLES COLLECTION AND PREPARATION

The samples were obtained at six (6) different local restaurants within Maiduguri Metropolitan

Council (MMC), comprising of Borno State University, Borno Express, Tanker Park, Post Office,

Tashan Kano serving as study samples and UMTH serving as control sample. Placed on a buffer

solution (peptone water) into the universal sterile containers, all samples were placed into

different sterile labeled containers. The samples were brought to the bacteriology Laboratory in

the Department of Microbiology, University of Maiduguri Teaching Hospital for further analysis.

3.4 CULTURING METHOD OR SAMPLE INNOCULATION

The samples were subjected to culture as well as sub-cultures.

Aseptically aided by biosafety cabinet, using a sterile inoculating loop, the innoculum was

streaked into TCBS agar (a selective medium for the isolation of V. cholerae and V.

parahaemolyticus as well as other Vibrio species), MacConkey Agar (a differential medium for

lactose and non-lactose fermenting organism) to check the growth of Escherichia coli and

coliform respectively. The streaked plates were incubated in an inverted form at 37°C for

24hours in the incubator.

3.6 Biochemical Tests

3.6.1 INDOLE TEST

The indole test was performed using peptone water. Indole test is a biochemical test performed

on bacterial species to determine the ability of the organism to convert tryptophan to indole using

the enzyme, tryptophanase.

34
The pure cultures were grown in a sterile SIM medium for 24hours, 2 drops of Kovacs reagent

were added to the medium. A positive result is shown by presence of red-violet colour on the

surface layer of the broth.

3.6.2 UREASE TEST

The test is used to differentiate organisms based on their ability to hydrolyze urea with the

enzyme urease.The enzyme urease leads to the breakdown of the urea to ammonia (NH3) and

carbon dioxide (CO2).

The pure cultures were streaked on the agar slant then the medium was incubated at 37°C for

24hours leaving the cap on loosely.

3.6.3 CITRATE UTILIZATION TEST

The test is done to determine the ability of bacteria to utilize Citrate as its only carbon source.

The pure cultures were inoculated on the Simmons Citrate agar slant and then incubated at 37°C

for 24 hours. A change in color of the media from green to blue is indicative of a positive result

while no color change indicates a negative result.

35
 CHAPTER FOUR

4.0 RESULT

The samples analyzed for vibrio, E.coli, and other coliform contamination were obtained from
Borno State University labeled(A) Borno Express labeled(B), Tanker Park labeled(C), Post
Office labeled(D), Tashan Kano labeled(E), as well as University of Maiduguri Teaching
Hospital (UMTH) labeled(F), revealed the following results.

Table: 4.1 shows the Initial culture (growth) of bacteria of MacConkey agar, while growth of
L.F was observed in samples labeled, A, B, and C while D, E, and F control showed no L.F
growth.

Table: 4.1 INITIAL CULTURE OF MACCONKEY AGAR

_____________________________________________________________________________________
Sample code Growth
A Lactose Fermentors
B Lactose Fermentors
C Lactose Fermentors
D No Bacterial Growth
E No Bacterial Growth
F No Bacterial Growth
__________________________________________________________

Table: 4.1.1 shows L.F growth and NBG. Three samples out of (5) showed 60% L.F
growth while two samples showed 40% NBG.

Table: 4.1.1 FREQUENCY AND PERCENTAGE

_____________________________________________________________________________________
S/N BG Frequency Percentage
1 LF 3 60%
2 NBG 2 40%
__________________________________________________________________
5 100%
_____________________________________________________________________________

36
4.2 BIOCHEMICAL TESTS
The below shows the Biochemical test results obtained after Subculture on McConkey agar.

Table: 4.2
_____________________________________________________________________________
Sample code citrate test indole test urease test inference
_____________________________________________________________________________
A Negative positive negative Escherichia coli

B Positive negative positive Klebsiella

C Positive positive positive Coliform

_____________________________________________________________________________

4.3 INITIAL CULTURE ON TCBS.

Table: 4.3 shows the growth of Vibrio spp on Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS
Agar) which shows presence of vibrio in samples B and C , while sample A, D, E and control
shows no bacterial growth.

Table: 4.2 INITIAL CULTURE ON TCBS.

______________________________________________________________________________
Sample code growth
______________________________________________________________________________
A No Bacterial Growth

B Yellow colonies

C Yellow colonies

D No Bacterial Growth

E No Bacterial Growth

F No Bacterial Growth

37
Table 4.3.1 show yellow colonies which indicate presence of vibrio in 40% of the samples, while
NBG is 60%.

Table 4.3.1
_____________________________________________________________________________________
S/N BG Frequency Percentage
1 Yellow Colonies 2 40%
2 NBG 3 60%
__________________________________________________________________
5 100%
_____________________________________________________________________________

Table 4.4 shows colonies grown by Subculture, of TCBS Agar, where samples B and C showed
presence of large yellow colonies of vibrio cholorae

Table: 4.4 SUBCULTURE ON TCBS.

__________________________________________________________________
Sample Colony Organisms
__________________________________________________________________
B Large yellow colonies Vibro Cholerae

C Large yellow colonies Vibro cholera

__________________________________________________________________

Table (4.5) describes the relationship between the samples containing the presence of both L.F,
Vibrio, and NBG.
__________________________________________________________________
S/N Sample L.F V. C
__________________________________________________________________
1. A L.F NBG
2. B L.F V.C
3. C L.F V.C
4. D NBG NBG
5. E NBG NBG
________________________________________________________________

38
 CHAPTER FIVE

5.1 DISCUSSION

Bacterial contamination of cooked food poses a serious health risk to consumers and can lead to
foodborne illnesses. It's crucial to explore various factors that contribute to bacterial
contamination, such as improper food handling, storage, and inadequate cooking temperatures.
Salmonella, E. coli, shegella, and Klebsiella, can provide valuable insights into the potential
health consequences and symptoms associated with these pathogens. it's vital to highlight the
importance of implementing good hygiene practices in both domestic and commercial kitchens,
as well as emphasizing the significance of thorough cleaning and sanitation of food preparation
surfaces, equipment, and utensils.

Findings from this study show that considerably some of the commonly consumed cooked food
in this study area, are contaminated with a wide variety of bacteria, which may pose serious
health risks and can lead to foodborne illnesses, such as gastrointestinal infections, fever,
diarrhea, nausea, abdominal pain, vomiting, and even more serious complications.in this research,
the bacteriological analysis was conducted and the study revealed that in Table ( 4.1) growth of
L.F was observed in samples labeled, A, B, and C while D, E, and control showed no L.F growth.

In this research, the bacteriological analysis conducted and the study revealed that in Table (4.1.1)

60% percent (3) of it samples are contaminated with lactose fermentors (L.F) and this findings is

in concordance with similar studies in Bangladesh around schools in Dhaka, by Al Mamun,

Rahman and Turin (2013) where nearly half of the samples (44.5%) percent were

contaminated with lactose fermentors (L.F).

In Table (4.2) the Biochemical analysis of pure isolate was revealed in three different samples. In

sample (A) citrate test shows Negative, the indole test is positive, and the Urease is Negative. In

sample (B) citrate test shows positive, the indole test is Negative and the Urease is positive.

39
Finally, in sample (C) citrate test shows positive, indole tested positive, and also Urease indicates

positive.

In Table (4.3.1),40% percent (2) of the sample was contaminated with vibrio cholerae and this

conforms with several studies conducted by many researchers, in an attempt to determine or

identify the microbial population responsible for cooked spoilage.

Table (4.5) describes the relationship between the samples containing the presence of both L.F,

Vibrio, and NBG.

5.2 CONCLUSION

At the end of this research work, the result from selected restaurants gives significant growth on,

MacConkey agar coliform is identified, while thiosulfate Citrate Bile Salts Sucrose Agar (TCBS

Agar) determined Vibrio spp. respectively.

The isolated organisms were identified by their morphological characteristics and other

biochemical’s such as Catalase, Coagulase and Urease test.

Indole test is a biochemical test performed on bacterial species to determine the ability of the

organism to convert tryptophan to indole using the enzyme, tryptophanase.

Citrate utilizing test, the test is done to determine the ability of bacteria to utilize Citrate as its

only carbon source.

Urease test, the test is used to differentiate organisms based on their ability to hydrolyze urea

with the enzyme urease. This biochemical's tests result were reported based on charges either

positive or negative.
40
5.3 RECOMMENDATIONS

1. Timely washing and replacement of cutting boards can decrease the possibility of biofilm

formation and cross-contamination.

2. Proper general hygiene, kitchen design and sanitation and cleaning methods according to

scientific instruction should be considered as the main principle to reduce contamination of

food contact surfaces.

3. Workers need to have a general hygiene certificate and participate in periodic training

courses according to the University’s schedule.

4. Also, continuous training, development of standards, more intensive supervision,

modification of behavior, designing food producing units in the University according to

scientific principles and implementation of food safety management should be considered by

the University authorities.

5. creating awareness campaign on safe and hygiene.

41
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APPENDIX

Lactose Fermentor (LF)

Escherichia Coli (E.coli)

Ready-To Eat (RTE)

Gastrointestinal Tract (GIT)

Centre For Disease Control (CDC)

World Health Organization (WHO)

NO Bacteria Growth (NBG)

Vibrio Cholerae (VC)

Thiosulfate-Citrate Bile Salts Sucrose (TCBS)

PREPARATION OF CULTURE MEDIA

All the culture media were prepared according to the manufacturers Standard Instructions. For
Thiosulfate citrate bile salts sucrose agar (TCBS agar),22.5g in 250ml 0f distilled water was
Mixed to dissolved, the mixture was heated on a Bunsen burner, Once boiling, continue heating
for another 1-2 minutes, stirring continuously to prevent the agar from burning, Once sterilized,
TCBS agar is poured into sterilized petri dishes and allowed to set. The petri dishes are covered
to prevent contamination. Once the agar has solidified, it is ready for use.

TCBS agar, is a type of selective agar culture plate that is used in microbiology laboratories to
isolate Vibrio species. TCBS agar is highly (selective for the isolation of V. cholera and V.
parahaemolyticus as well as other Vibrio species).

The MacConkey Agar was prepared using 12g in 250ml of distilled water and autoclaved for
15mintutes, brought out to attain room temperature, afterward it was distributed to various Petri
dishes, where it solidified later as it contains solidifying agent called Agar-Agar. And all
Samples Were cultured on MacConkey Agar (MAC).

The samples were also cultured using a sterile inoculating loop by the streak plate method on
TCBS agar (a selective medium for the isolation of V. cholerae and V. parahaemolyticus as well
as other Vibrio species), MacConkey Agar (a differential medium for lactose and non-lactose
fermenting organism) to check the growth of Escherichia coli and coliform respectively. The
streaked plates were incubated in an inverted form at 37°C for 24hours in the incubator.

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All the above procedures were carried out aseptically to avoid possible contamination which
might interfere with the results.

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