BACTERIOLOGICAL QUALITY OF FROZEN CHICKEN
SOLD IN LAGOS METROPOLIS
BY
OKAFOR RITA NNENNA
P/ND/19/3710143
DEPARTMENT OF SCIENCE LABORATORY TECHNOLOGY
SCHOOL OF TECHNOLOGY
YABA COLLEGE OF SCIENCE
YABA-LAGOS
AUGUST, 2022
i
BACTERIOLOGICAL QUALITY OF FROZEN CHICKEN
SOLD IN LAGOS METROPOLIS
BY
OKAFOR RITA NNENNA
P/ND/19/3710143
IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE
AWARD OF NATIONAL DIPLOMA CERTIFICATE IN SCIENCE
LABORATORY TECHNOLOGY
TO
DEPARTMENT OF SCIENCE LABORATORY TECHNOLOGY
SCHOOL OF TECHNOLOGY
YABA COLLEGE OF SCIENCE
YABA-LAGOS
AUGUST, 2022
i
CERTIFICATION
This Thesis entitled ‘‘BACTERIOLOGICAL QUALITY OF FROZEN CHICKEN SOLD IN
LAGOS METROPOLIS’’ by Okaafor, Rita Nnenna, meet the regulation governing the award of
the degree of National Diploma of Yaba College of Technology, Yaba, Lagos and is approved for
its contribution to scientific knowledge and literary presentation.
Mr. Famakinwa, O.A
Date
Project supervisor
Mr. Aderibigbe, T. A
Date
Coordinator
Dr. (Mrs.) Popoola, E. O.
Date
Head of department
ii
DECLARATION
We hereby declare that this Thesis has been written by me and is a record of my own research
work. It has not been presented in any previous application for a higher degree of this or any other
institution. All citation and sources are clearly acknowledged by means of references.
……………………………………
Okafor Rita Nnenna
Date…………………………
iii
DEDICATION
This project is dedicated to almighty God who made me to be what I am today and to be involved
in such a marvelous project. To him are all Glory, Honour and Adoration, now and forever, Amen.
iv
ACKNOWLEDGEMENT
With a grateful heart my utmost thanks goes to ALMIGHTY GOD for bringing us this far and
being a source of wisdom and understanding.
To our supervisor, Mr. Famakinwa, O.A I say thank you for believing in me to the end and for
your great inspiration, motivation and encouragement and my appreciation goes to all my lecturers.
I would also like to express my sincere appreciation to my parent, my entire family and friends,
for their support and encouragement.
I will also appreciate the support and hard work of my group member, god bless you all.
v
ABSTRACT
Poultry production is dominated by chicken in many parts of the world due to the relative ease of
its production. The aim of the study is to determine the bacteriological quality of frozen chicken
sold in Lagos metropolis. Principle test the ability of an organism to utilize citrate as its sole carbon
and energy source for growth and ammonium salt as the source of nitrogen with resulting
alkalinity. Twenty samples of frozen chicken from seven different sellers were analyzed to
determine their bacteriological load; the samples were collected from Ijora, Sabo, Mushin, Bariga,
Somolu, Oyingbo and Onipanu market Lagos state. All the frozen chicken samples from the seven
sellers in different markets examined were contaminated with some bacterial species namely,
Bacillus cereus, Escherichia coli, Staphyloccus aureus, Staphylococcus epidermidis, Proteus
vulgaris, Klebsiella pneumonia, Pseudomonas aeruginosa. The total bacteria count for all the
chicken examined from the different sellers was in the range of 0.7 × 102 cfu/ml to 8.5 × 102 cfu/ml
and the coliform counts obtained for all the chicken samples ranged from 0.1 × 102 cfu/ml to 3.2
× 102 cfu/ml. These finding suggest that most of the frozen chicken parts stored in the open market
may constitute sources of bacterial food poisoning consequently public health hazard. It is
concluded that a good personal and environmental hygiene should be put in place in other to reduce
the high level of microbial load recorded in some of the location
vi
TABLE OF CONTENT
Page
Title page
1
Declaration
ii
Certification
iii
Dedication
iv
Acknowledgement
v
Abstract
vi
Table of content
vii
List of tables
vii
List of figures
ix
CHAPTER ONE: INTRODUCTION
1.1
Background to the Study
1
1.2
Aims and Objectives
4
CHAPTER TWO: LITERATURE REVIEW
2.0
Introduction
5
2.1
Concept of Chicken
5
2.2
Nutritional Value of Chicken
6
2.3
Uses of Chicken
7
2.4
Foodborne Organisms Associated with Chicken
9
2.5
Factors Influencing Chicken Contamination
14
2.6
Control of Chicken Contamination
15
vii
CHAPTER THREE: METHODOLOGY
3.1
Sample collection
19
3.2
Cleaning and sterilization of materials
20
3.3
Media preparation
21
3.4
Dilution of sample
21
3.5
Microbial load determination
21
3.6
Isolation of pure cultures
21
3.7
Characterization and identification of isolates
22
3.8
Biochemical identification
23
3.9
Citrate utilization test
24
CHAPTER FOUR:
4.1
Result
26
CHAPTER FIVE
5.1
Discussion
34
5.2
Conclusion
35
viii
LIST OF TABLES
Table 2.1
Nutritional contents of chicken
7
Table 4.1
Morphological characteristics of isolated bacteria
26
Table 4.2
Total microbial load count of the various samples
30
Table 4.3
Gram stainiging and biochemical identification of the isolated bacteria
32
ix
CHAPTER ONE
INTRODUCTION
1.1
Background to the Study
Poultry production is dominated by chicken in many parts of the world due to the relative ease of
its production (Mottet & Tempio, 2017) and serve as a major source of animal protein (Cadudal,
2017). Meanwhile, chicken meat is known to cause many zoonotic foodborne infections in the
world (Rouger, Tresse & Zagorec, 2017). Global food production and consumption systems,
therefore, required that food quality and safety be scrutinized and examined for public health safety
(Wahyono and Utami, 2018; Baltic, 2019). Poultry and poultry meat are however prone to
contaminations with potentially pathogenic microorganisms such as Salmonella, Campylobacter,
S. aureus, E. coli and Listeria (Castañeda-Gulla, Sattlegger, Mutukumira, 2020)
Generally, the microbial quality of meat products including chicken has purchased by consumers
depends on factor such as the quality of raw products and other materials used or added during
processing operations to the product has extraneous contaminants; efficacy of cooking process;
sanitation during processing and packaging; maintenance of adequate refrigeration from the
processing to the retail level and to the consumer; and finally sanitation during handling at the
retail end (Selvanet al., 2007). Poultry meat offers an excellent medium for the multiplication of
most bacteria, including those that are not inhibited by lowtemperatures, storage of processed
poultry meat is vita and therefore considered only under circumstance which inhibit the
multiplication of the initial load of bacteria
Food security is a complex issue, where animal proteins such as meats, meat products, fish, chicken
and fishery products are generally regarded as high risk commodity in respect of pathogen
1
contents, natural toxins and other possible contaminants and adulterants (Yousuf et al., 2008).
Food borne infections and illnesses is a major international health problem with consequent
economic reduction. It is a major cause of illness and death worldwide (Adak et al., 2005).
Recognizing this, the World Health Organization (WHO) developed its Global Strategy for Food
Safety (Adak et al., 2005). In the developing world, foodborne infection leads to the death of many
children and the resulting diarrheal disease can have long-term effects on children's growth as well
as on their physical and cognitive development (Adak et al., 2005). In the industrialized world,
foodborne infection causes considerable illness, heavily affecting healthcare systems (Adak et al.,
2005). According to Clarence et al., (2009), food borne diseases are diseases resulting from
ingestion of bacteria, toxins and cells produced by microorganisms present in food. The intensity
of the signs and symptoms may vary with the amount of contaminated food ingested and
susceptibility of the individuals to the toxin (Clarence et al., 2009).
Chicken the most perishable of all important foods since it contains sufficient nutrient needed to
support the growth of microorganisms (Magnus, 1981). The chief constituents of meat are water,
protein and fat, phosphorus, iron and vitamins are also contained in meat. The major primary unit
of meat is called carcass. It represents the ideal meat after head, hide, intestine, blood. The edible
parts of a carcass include lean flesh, fat flesh and edible glands or organs such as heart, liver,
kidney tongue and brain. Meat is considered as the most nutritive source of protein consumed by
humans. Age and sex of the animal has a major influence on the quality of meat that is produced
from animals (Rao et al., 2009). Most meat have high water content corresponding to the water
activity approximately 0.99 which is suitable for microbial growth (Rao et al., 2009)
Foodborne microbiologic hazards may be responsible for as many cases of illness as possible each
year and are thus an important food safety challenge. To lower the incidence of foodborne disease,
2
many experts and stakeholders urge the development of a science- and risk-based food safety
system, in which decision makers prioritize hazards and interventions using the best available data
on the distribution and reduction of risks (Batz et al., 2005). Such a system requires an
understanding of the many risk factors between the point of production and the point of
consumption and the ability to systematically target intervention efforts along this "farm-to-fork"
continuum (Batz et al., 2005). The preservation of meat as a perishable food usually is
accomplished by a combination of preservation methods which greatly lengthen the keeping
quality the meat. So, to increase meat quality assurance in accordance with microbial load
assessment is deemed necessary (Yousuf et al., 2008).
1.2
AIMS AND OBJECTIVES
The aim of the study is to determine the bacteriological quality of frozen chicken sold in Lagos
metropolis.
In order to achieve this aim, the following objectives are stated:
i.
To isolate the various microbial isolates associated with frozen chicken purchased from
different sellers in Yaba market, Lagos.
ii.
To characterize and identify these micro-organisms.
iii.
To speculate on the significance of these isolates.
iv.
To compare the level of contamination of the samples (frozen chicken) collected from
different parts of the market.
3
CHAPTER TWO
LITERATURE REVIEW
2.0
Introduction
Increased consumption of poultry has resulted in an increase of poultry-associated food borne
disease, particularly salmonellosis. Poultry ranks first or second in foods associated with disease
in Australia, Canada, England and Wales and fourth in the United States.
2.1
Concept of Chicken
The chicken (Gallus gallusdomesticus) is a domesticated fowl, a subspecies of the Red Junglefowl,
first domesticated in India around 2000 B.C. (Wong, 2004). As one of the most common and
widespread domestic animals, and with a population of more than 24 billion in 2003, there are
more chickens in the world than any other species of bird (Wong, 2004). The domestic chicken is
descended primarily from the Red Junglefowl (Gallus gallus) and is scientifically classified as the
same species (Wong, 2004). Chickens are omnivores (Grandin and Johnson, 2005) in the wild,
they often scratch at the soil to search for seeds, insects and even larger animals such as lizards or
young mice (Worrell, 2008). Chickens may live for five to ten years, depending on the breed. The
world's oldest chicken, a hen, died of heart failure at the age of 16 according to the Guinness Book
of World Records (Smith, 2006). Broiler-fryers, roasters, stewing/baking hens, capons, and Rock
Cornish hens are all chickens. The following are definitions for these:
•
Broiler-fryer – a young, tender chicken; about 7 weeks old; weighs 2½ to 4½ pounds when
eviscerated.
•
Rock Cornish Game Hen – a small broiler-fryer; weighs between 1 and 2 pounds.
•
Roaster – an older chicken; about 3 to 5 months old; weighs 5 to 7 pounds. It yields more
meat per pound than a broiler-fryer.
4
•
Capon – male chickens; about 16 weeks to 8 months old; surgically unsexed. They weigh
about 4 to 7 pounds and have generous quantities of tender, light meat.
•
Stewing/Baking Hen – a mature laying hen; 10 months to 1½ years old.
•
Cock or rooster – a mature male chicken with coarse skin and tough, dark meat.
2.2
Nutritional Value of Chicken
Chicken is rated as a very good source of protein, providing 67.6% of the daily value for protein
in 4 ounces. The structure of humans and animals is built on protein. We derive our amino acids
from animal and plant sources of protein, and then rearrange the nitrogen to make the pattern of
amino acids we require. The table below shows the percentage daily value that a serving of chicken
for each of the nutrients of which it is a good, very good or excellent source.
TABLE 2.1: NUTRITIONAL CONTENTS OF CHICKEN
Nutrients in Chicken 4.00 oz-wt (113.40 grams)
Nutrient
% Daily Value
Tryptophan
128.1
Vitamin B3
77.7
Protein
70.3
Selenium
44.7
Vitamin B6
34
Phosphorus
25.8
Choline
22.7
Calories (187)
10
Source: Castañeda-Gulla, Sattlegger, Mutukumira, 2020
5
2.3
Uses of Chicken
Humans keep chickens primarily as a source of food, consuming both their meat and their eggs.
The chicken's "cultural and culinary dominance" could be considered amazing to some in view of
its believed domestic origin and purpose and it has "inspired contributions to culture, art, cuisine,
science and religion" (Adler and Lawler 2012) from antiquity to the present. The traditional poultry
farming view of the domestication of the chicken is stated in Encyclopedia Britannica (2007):
"Humans first domesticated chickens of Indian origin for the purpose of cockfighting in Asia,
Africa, and Europe. Very little formal attention was given to egg or meat production... "(Garrigus,
2007)
1
Reared for meat
Chickens (broiler) farmed for meats are called broiler chickens. Chickens will naturally live for 6
or more years, but broiler chickens typically take less than 6 weeks to reach slaughter size
(Fumihitoetal., 1994). A free range or organic meat chicken will usually be slaughtered at about
14 weeks of age.
2
Reared for eggs
Chickens farmed for eggs are called egg-laying hens. Hens do not need a male to produce eggs,
only to fertilize them. A flock containing only females will still produce eggs, however the eggs
will all be infertile (Glenday, 2011). In 2009, an estimated 62.1 million metric tons of eggs were
produced worldwide from a total laying flock of approximately 6.4 billion hens.
3
Reared as food
Eggs - Chicken eggs are widely used in many types of dishes, both sweet and savory, including
many baked goods. Eggs can be scrambled, fried, hard boiled, soft-boiled, pickled, and poached.
The albumen, or egg white, contains protein but little or no fat, and can be used in cooking
6
separately from the yolk. Ground egg shells are sometimes used as a food additive to deliver
calcium.
Chicken - The meat of the chicken, also called "chicken", is a type of poultry meat. Because of its
relatively low cost, chicken is one of the most used meats in the world. Nearly all parts of the bird
can be used for food, and the meat can be cooked in many different ways. Popular chicken dishes
include roasted chicken, fried chicken, chicken soup, Buffalo wings, tandoori chicken, butter
chicken, and chicken rice. Chicken is also a staple of many fast food restaurants.
4
Reared as Pets
Chickens are sometimes kept as pets and can be tamed by hand feeding, but roosters can sometimes
become aggressive and noisy, although aggression can be curbed with proper handling. Some have
advised against keeping them around very young children. Certain breeds, however, such as silkies
and many bantam varieties are generally docile and are often recommended as good pets around
children with disabilities. Some people find chickens' behaviour entertaining and educational
(Glenday, 2011).
2.4
Foodborne Organisms Associated with Chicken
Food security is a complex issue, where animal proteins such as meats, meat products, fish and
fishery products are generally regarded as high risk commodity in respect of pathogen contents,
natural toxins and other possible contaminants and adulterants (Yousuf et al., 2008). Food borne
infections and illnesses is a major international health problem with consequent economic
reduction. It is a major cause of illness and death worldwide (Adak et al., 2005). Recognizing this,
the World Health Organization (WHO) developed its Global Strategy for Food Safety (Adak etal.,
2005). In the developing world, foodborne infection leads to the death of many children and the
resulting diarrheal disease can have long-term effects on children's growth as well as on their
7
physical and cognitive development (Adak et al., 2005). In the industrialized world, foodborne
infection causes considerable illness, heavily affecting healthcare systems (Adak et al., 2005).
According to Clarence et al., (2009), food borne diseases are diseases resulting from ingestion of
bacteria, toxins and cells produced by microorganisms present in food. The intensity of the signs
and symptoms may vary with the amount of contaminated food ingested and susceptibility of the
individuals to the toxin (Clarence et al., 2009). Foodborne microbiologic hazards may be
responsible for as many cases of illness as possible each year and are thus an important food safety
challenge. To lower the incidence of foodborne disease, many experts and stakeholders urge the
development of a science- and risk-based food safety system, in which decision makers prioritize
hazards and interventions using the best available data on the distribution and reduction of risks
(Batz et al., 2005). Such a system requires an understanding of the many risk factors between the
point of production and the point of consumption and the ability to systematically target
intervention efforts along this "farm-to-fork" continuum (Batz et al., 2005).
Most foodborne illness outbreaks are a result of contamination from food handlers. Sanitary food
handling and proper cooking and refrigeration should prevent foodborne illnesses. Bacteria must
be consumed on food to cause foodborne illness. They cannot enter the body through a skin cut.
However, raw poultry must be handled carefully to prevent cross-contamination. This can occur if
raw poultry or its juices come in contact with cooked food or foods that will be eaten raw, such as
salad. An example of this is using a cutting board to chop raw chicken and then using the same
board to chop tomatoes without washing the board first.
8
2.4.1 Salmonella and Campylobacter
Contamination of poultry carcasses and parts with these organisms is well documented and data
are available for many parts of the world (Waldroup, 1996), although inter-country comparisons
are not usually possible, because of differences in sampling and methods of testing. Most
salmonellas found on poultry meat are non-host- specific and are considered capable of causing
human food poisoning. The thermophilic campylobacters are mainly Campylobacter jejuni, which
is the principal cause of human campylobacteriosis, but other, so-called campylobacteria also
occur frequently, and include species of Acrobacter and Helicobacter pullorum. According to
Corry and Atabay (2001), both types of bacteria have the potential of causing human illness and
they both include strains that are invasive in poultry and can penetrate internal organs or deep
tissues of the bird, where the organisms may be less readily destroyed by cooking. Since the
infective dose is only a few hundred viable cells, illness can easily result from handling raw poultry
without suitable hygiene precautions, and is a potential hazard for new staff in poultry processing
plants. Campylobacter jejuni is one of the most common causes of diarrheal illness in humans,
preventing cross-contamination and using proper cooking methods reduces infection by this
bacterium.
Salmonellas survive well in the environment, but campylobacters appear less well-adapted to
survival outside the alimentary tract of warm-blooded animals. Also, growth only occurs under
conditions of high moisture, reduced oxygen and an environmental temperature above 30 ºC. The
organisms are particularly sensitive to drying and the effects of freezing and thawing, which can
cause a 1 - 2 log reduction in the level of contamination on poultry meat. However, campylobacters
have many different hosts, they colonise at high levels and therefore are shed into the environment
in large numbers. There is still much debate about possible survival mechanisms outside the host,
9
including the ability to exist in a supposedly dormant form, in which the organisms appear to be
viable, but non-culturable by conventional methods. From the practical viewpoint, campylobacters
can persist as contaminants of poultry products throughout the entire supply chain and remain
detectable by cultural methods. A key factor in their survival may be their attachment to, or
entrapment in, poultry tissues during carcass processing. In this situation, their resistance to
adverse conditions, like that of other bacteria, is significantly increased. Thus, the organisms can
survive on carcasses during processes such as scalding, washing and water chilling, that might
otherwise remove or destroy them (USDA, 2012).
2.4.2 Clostridium perfringens
As a cause of human food poisoning, this is not among the more dangerous pathogens. It is,
however, a spore-forming organism and some strains produces spores that are unusually heatresistant. Therefore, unlike vegetative bacterial cells, the spores are not necessarily destroyed by
normal cooking and may subsequently germinate and outgrow to hazardous levels, if post-cooking
storage is inadequate. In fact, most outbreaks involve strains that produce the more heat-resistant
spores. The organism is an obligate anaerobe that is relatively tolerant to oxygen and can be found
in low numbers in the alimentary tract of poultry. When present in meat crevices etc, growth is
favoured by conditions in which oxygen has been dispelled by cooking. However, since growth of
the organism cannot occur if the meat is held below 15 ºC, the problem is easily avoided by
refrigerated storage (USDA 2012).
2.4.3 Escherichia coli
Escherichia coliis a type of bacteria that normally lives in the intestines of animals and humans.
There are hundreds of different kinds, or strains, of E. coli, some of which can be harmful, but
10
most are not. Animal meats may become contaminated with this bacterium during the slaughter
process (USDA, 2012).
The presence of E. coli, although an indicator organism for faecal matter, does not mean the
product is, in fact, contaminated by faeces. E. coli that is present in feathers or environmental
contaminants, like dust, can also contaminate a poultry carcass. E.coli causes diarrhoea and
haemorrhagic colitis in humans and can lead to potentially life-threatening sequelae, such as
haemolyticuraemic syndrome and thrombotic thrombocytopaenicpurpura (USDA, 2012).
2.4.4 Listeria monocytogenes
The organism is a leading cause of food-related mortality and morbidity in man, and the majority
of cases are believed to be foodborne. The symptoms vary widely and those affected are frequently
among the most vulnerable groups in society. Listeria monocytogenes was recognized as causing
human foodborne illness in 1981. It is destroyed by cooking, but a cooked product can be
contaminated by improper handling or poor sanitary practices in food preparation and storage
areas. The risk from Listeria monocytogenes can increase when it has the opportunity to grow on
a food product in storage. The organism is common on raw poultry meat and has been found on
chicken, turkey, duck and pheasant. Numerous surveys have shown that more than 50% of
processed chicken carcasses are likely to be positive, although numbers are usually low, even < 1
/ cm2 of skin.
The health hazard from contaminated, raw poultry is mainly one of cross-contamination in the
kitchen, where the organism may spread to cooked foods or other ready-to-eat items, such as salad
vegetables. There is also a potential problem with cooked poultry produced commercially.
Although normal cooking destroys listerias, recontamination can occur during post-cooking
11
handling at the factory, even with the most rigorous hygiene control. Since pre-cooked items are
not necessarily reheated by consumers before being eaten, and the organism is capable of growth
under chill conditions, strict microbiological limit values are considered necessary (USDA, 2012).
2.4.5 Spoilage organisms
When poultry meat is stored aerobically under chill conditions, the organisms that predominate at
spoilage are invariably Pseudomonas spp., accompanied by lower numbers of other Gram-negative
bacteria. Using numerical taxonomy, Arnaut-Rollieretal., (1999) found four major clusters of
pseudomonads: Pseudomonas fragi, Pseudomonas lundensis, Pseudomonas fluorescensbiovars
and an unidentified group resembling Pseudomonas fluorescens. Other bacteria that are sometimes
present include Shewanellaputrefaciens and psychrotrophic strains of Enterobacteriaceae. The
above organisms are common in soil and water, and are thought to originate from the live-bird
environment.
At spoilage, the predominant organisms are usually lactic acid bacteria, but other Gram-positive
and Gram-negative bacteria can occur in relatively large numbers.
2.5
Factors Influencing Chicken Contamination
Companies are able to prevent contamination by properly rotating their stock. Product that is to be
sold in locations far from the processing plant should be transported at temperatures that are below
freezing (i.e. 26 F), but not sufficient to freeze the muscle tissue (deep chill). Inappropriate storage
temperatures or fluctuations in storage temperature are the most avoidable causes of
contamination. Temperature abuse can occur during distribution, storage, retail display or handling
of the product by the consumer. Processors can determine whether product has been temperature
12
abused by monitoring temperature or evaluating bacterial populations throughout the distribution
system (USDA, 2012).
Initial bacterial counts on broiler carcasses may have a direct effect on the shelf-life of fresh
product as well. The initial number of bacteria on poultry is generally a function of grow-out
procedures, production practices, and plant and processing sanitation. Higher numbers of spoilage
bacteria on the chicken immediately after processing, translates to more rapid contamination. High
post-rigor meat pH is often caused by stress on the birds during grow-out or transportation. This
reduces the shelf-life of the meat by up to six days and is due to the fact that spoilage bacteria
multiply much more rapidly on meat that is at a pH of 6.2 than on meat that is at a normal postrigor pH of 5.4-5.6.
Understanding how poultry products become contaminated is critical to finding ways to detect and
prevent spoilage. The primary causes of poultry products contamination are as follows:
•
Prolonged distribution or storage time
•
Inappropriate storage temperature
•
High initial bacterial counts
•
High post-rigor meat pH
•
Dealing with spoilage factors
As on any perishable meat, fish, or poultry, bacteria can be found on raw or undercooked chicken.
They multiply rapidly at temperatures between 40 and 140 °F (4.4 and 60 °C) — (out of
refrigeration and before thorough cooking occurs). Freezing doesn't kill bacteria, but they are
destroyed by thorough cooking. These four food safety steps will keep chicken safe from food
borne bacteria.
13
2.6
•
Clean: Wash hands and surfaces often.
•
Separate: Separate raw meats and poultry from other foods.
•
Cook: Cook all poultry to 165 °F (73.9 °C).
•
Chill: Refrigerate promptly.
Control of Chicken Contamination
For food to be entirely safe from the microbiological viewpoint, it would need to be free from all
pathogenic organisms. It is widely recognised, however, that this is not a realistic goal for raw
poultry meat. There is still no economically viable means of eliminating foodborne pathogens in
poultry-meat production, without the use of ionising radiation, which is presently unacceptable to
many consumers. Therefore, some level of product contamination must be tolerated, although this
varies widely from one country to another, especially in relation to Salmonella. In Sweden, which
has a small poultry industry, the prevalence of Salmonella- contaminated poultry meat has been
less than 1% for many years and the organisms are rarely found in retail samples due to rigorous
surveillance and control programmes that are relatively costly to operate (Persson and Jendteg,
1992). Food from which salmonellas are isolated in Sweden is, by law, considered unfit for human
consumption.
The microbiological hazards in the processing operation are well known and are often difficult to
control effectively, because of the technological limitations in the process that can lead to crosscontamination of the carcasses being processed. Implementation of the HACCP system does not
overcome this drawback, but has a number of clear benefits, including the following:
1. The system ensures regular monitoring of the process as a whole.
2. Hygiene control is optimised, within the above-mentioned constraints, thereby providing
evidence of due diligence on the part of the processor, as required by UK food law.
14
3. Checking of control parameters and recording of results are an integral part of the system.
4. Compliance with hygiene legislation is ensured.
5. Staff awareness of food-safety requirements is increased.
6. As a result of national HACCP implementation, operational standards across the Industry
become more uniform.
Although use of the HACCP system in poultry processing is aimed primarily at the control of
foodborne pathogens, there is also the potential to reduce contamination of carcasses with spoilage
organisms. Pseudomonads, in particular, are largely destroyed during scalding and carcasses are
re- contaminated during subsequent stages of processing (Mead, 2004). It is these stages that need
to be targeted for control purposes.
Cross-contamination of carcasses with pathogens can occur at virtually every stage of the process
and currently there is little evidence that this problem is significantly reduced by the application
of HACCP principles. Also unclear is the effect of the HACCP system on levels of carcass
contamination, although this will vary according to the type of process used and permitted
intervention measures in different countries. The most effective type of process for reducing
contamination is likely to be one in which carcasses are immersion-chilled in chlorinated water
and then frozen but because super-chlorination is not allowed, immersion chilling has been largely
replaced by air chilling or evaporative cooling, and any form of chemical decontamination is
unacceptable. Therefore, in the case of fresh carcasses that are air chilled, there is no marked
reduction in carcass contamination (Allen etal., 2000; Fluckeyetal., 2003). Moreover, there is no
Critical Control Point at which a significant reduction in pathogen contamination can be
guaranteed. However, this unsatisfactory situation may change in 2006 (Council of European
15
Union, 2003). Without the use of processing aids to improve hygiene, the greatest reductions in
carcass contamination are likely to come from technological developments in the process that are
designed to improve hygiene, as long as these are acceptable to the Industry. For example, a
process for simultaneous scalding and plucking of broilers, although not adopted commercially,
reduced levels of Enterobacteriaceae on carcasses by one hundred-fold in experimental trials
(Mulder, 1985).
16
CHAPTER THREE
METHODOLOGY
3.1
SAMPLE COLLECTION
The total of Eight (8) samples of frozen chicken parts were collected from five locations namely
Onyingbo, Ijora, Sabo, Mushin, Onipanu, Bariga and Somolu markets respectively using a sterile
zip-lock bags and an iced flask. The frozen chicken samples were first put in different zip-lock
bags and the individual samples were place inside an iced flask in other to maintain viability of
the bacteria cells as this was transported to microbiology Laboratory for an onward
microbiological analysis.
CULTURE MEDIUM USED FOR ISOLATION
•
Nutrient Agar (NA)
•
Eosin Methylene Blue Agar (EMB)
•
Salmonella Shigella Agar (SSA)
•
Manitol salt Agar (MSA)
•
Peptone water
•
Potato dextrose agar (PDA)
MATERIALS USED FOR SAMPLE COLLECTION
•
Zip-lock bag
•
Gloves
•
Iced pack
MATERIALS USED FOR INOCULATION
•
Cotton wool
•
Petri dishes
17
•
Inoculating loop
•
70% ethanol
•
Spirit lamp
•
Pipette
•
Test tube
•
Permanent marker
•
Masking tape
•
Gloves
•
McCartney bottles
•
Foil paper
•
Nose mask
EQUIPMENT
3.2
•
Autoclave
•
Incubator
•
Hot air oven
•
Weighing balance
•
Hot plate and magnetic stirrer
CLEANING AND STERILIZATION OF MATERIALS
All working glass wares were washed with soap and water; sterilized using the dry-heat method
(hot-air oven), at 1600C for 1hour, different media with moist heat method (autoclave) and the
work-bench with cotton wool soaked with 99.9% ethanol as the spirit-lamp was on to maintain an
aseptic environment.
18
3.3
MEDIA PREPARATION
All the media were prepared according to manufacturer’s manual and sterilized by autoclaving at
1210C for 15 minutes. The media were then allowed to cool to 450C before pouring into petri
dishes.
3.4
DILUTION OF SAMPLE
Samples were diluted using a 10-fold serial dilution in 7 steps techniques which involved weighing
1g of the frozen chicken in a mortar and blended using the pestle. Thereafter, each of the blended
samples were transferred into 9ml peptone water in a test tube as diluent, homogenized, labeled
tubes 1 to 7 as 10-1. 10-2. 10-3. 10-4. and 10-7 respectively and 1 ml were transferred to the next tube
10-2, homogenized and transferred 1ml to 10-3, 10-4 and 10-7 respectively as 1ml was discarded
from the last tube.
3.5
MICROBIAL LOAD DETERMINATION
The method of inoculation used was pour plate method as 0.1ml (aliquot) from dilution factor (104
) was plated out into a sterile petri-dishes and molten agar of Eosin methylene blue (EMB) agar,
Manitol salt agar (MSA), Salmonella shigella agar (SSA), Potato dextrose agar (PDA) and Nutrient
agar (NA) were poured aseptically, swirled and allowed to solidify followed by incubating at 370C
for 48hours for bacteria while fungi at 280C for 3days. The colonies were counted and the results
were expressed in colony forming unit for bacteria (cfu/g) and spore forming unit for fungi (sfu/g).
3.6
ISOLATION OF PURE CULTURES
After incubation, the isolates were enumerated and their morphological features noted. Each
isolate was sub-cultured three times to obtain pure cultures by streaking on a freshly prepared
19
nutrient agar medium. The plates were then incubated at 370C for 24hours. Each pure isolates were
thereafter transferred to a sterile nutrient agar slant which was then refrigerated for further analysis.
3.7
CHARACTERIZATION AND IDENTIFICATION OF ISOLATES
MORPHOLOGICAL CHARACTERISTICS
The morphological characteristics as a means of identification were carried out by determining
physical appearances of the isolate such as color, shape, edges (margin), elevation, and
consistency.
PATHOGENICITY TEST OF ISOLATES
The isolated bacteria were subjected to virulent test by preparing a blood agar using the standard
method which includes addition of 10mls of whole blood into a molten nutrient agar, homogenized
vigorously for complete mixture and then poured aseptically into fresh sterile petri dishes and was
allowed to solidify. Each representative isolates were inoculated on the blood agar using streaking
methods with the aid of a wire loop. Thereafter, incubated at 370C for 24hours as evidence of
lysing of the red blood cell in the plate by the production of zone of inhibition indicates POSITIVE
while the reverse case indicates NEGATIVE.
GRAM STAINING
Gram staining is a differential procedure use to differentiate bacterial into Gram positive and Gram
negative. After the staining procedure, the Gram positive bacteria retained their purple color after
counterstaining while the Gram negative bacteria loses its purple color and changes to pink or red.
20
3.8
BIOCHEMICAL IDENTIFICATION
The following biochemical tests were carried out on the isolates.
CATALASE TEST:
PRINCIPLE: This demonstrates the presence of catalase, an enzyme that catalysis the release of
oxygen from hydrogen peroxide.
PROCEDURE: 1ml of hydrogen peroxide solution was poured over a 24 hours nutrient agar slope
culture of the test organism and the tube held in a slanting position.
A loopful of the test organism was picked and dipped in a drop of 10 percent hydrogen peroxide
on a glass slide.
RESULT: Presence of immediate bubbles indicates POSITIVE while absence of bubbles indicates
NEGATIVE.
OXIDASE TEST
PRINCIPLE: To determine the presence of bacteria cytochromeoxidase using the oxidation of the
substrate, a redox dye, tetramethyl-p-phenylene-diamenedihydrochloride (oxidase reagent).
PROCEDURE: A drop of freshly prepared 1 percent solution of oxidase reagent was made on a
piece of filter paper. A colony of bacteria were picked using the inoculating loop and robbed on
the surface of the filter paper.
RESULT: Deep purple color within 10 seconds indicates POSITIVE while absence indicates
NEGATIVE.
UREASE TEST
PRINCIPLE: This is to detect the ability of an organism to produce an enzyme urease which splits
urea to carbon dioxide and ammonia.
21
PROCEDURE: The test organism was inoculated on the entire slope of christensen medium which
contain urea and phenol red indicator with other constituents including agar. It was incubated at
370C for 4days.
RESULT: Purple-pink color indicates POSITIVE while pale yellow color indicates NEGATIVE
NITRATE REDUCTION TEST
PRINCIPLE: This detects the presence of enzyme nitrate reductase which causes the reduction of
nitrate to nitrite.
PROCEDURE: The test organism was inoculated in a 5ml medium containing potassium
nitrate,peptone and disttlied water, incubated at 370C for 96 hours.
RESULT: Production of red color within few minutes indicates POSITIVE while absence
indicates NEGATIVE.
INDOLE TEST
PRINCIPLE: To detect the ability of bacteria to decompose amino acid tryptophan to produce
indole.
PROCEDURE: The test organism was inoculated in peptone water and incubated at 370 C for 96
hours. 0.5ml Kovacs reagent was added and gently shacked.
RESULT: Red color production indicates POSITIVE while yellow color ring indicates
NEGATIVE
3.9
CITRATE UTILIZATION TEST
PRINCIPLE: This test the ability of an organism to utilize citrate as its sole carbon and energy
source for growth and ammonium salt as the source of nitrogen with resulting alkalinity.
PROCEDURE: The test organism was inoculated in a simons citrate medium containing agar,
citrate and bromothymol blue as an indicator and incubated at 370 C for 96 hours.
22
RESULT: Blue color and growth of organism indicates POSITIVE while original green color and
no growth indicates NEGATIVE.
23
CHAPTER FOUR
RESULT
This table reveals the rate at which the various sample (frozen chicken) is contaminated
microbiological. The sample given some market in Lagos metropolis namely, Oyingbo, Ijora,
Sabo, Mushin, Bariga, Somolu and onions. The total microbial load using an all purpose
media(nutrient Agar)was conducted across all the sample from the various locations. It was
discovered that the sample from mushin yield the highest microbial load of 15.8×10 while The
lowest count was recorded at bargain with the load of 0.6×10
24
Table 4.1: Total microbial load count of the various samples
SAMPLE/LOCATION
MEDIA
DILUTION
NO. OF
TOTAL
FACTOR
COLONIES
MICROBIAL
LOAD (cfu/g)
ONYINGBO (A)
IJORA (B)
SABO (C)
MUSHIN (D)
44
4.4X10-5
EMB
07
0.7X10-5
MCA
NG
NG
SSA
NG
NG
MSA
NG
NG
PDA
06
0.6X10-5
30
3.0X10-5
EMB
02
0.2X10-5
MCA
NG
NG
SSA
NG
NG
MSA
NG
NG
PDA
04
0.4X10-5
32
3.2X10-5
EMB
01
0.1X10-5
MCA
NG
NG
SSA
NG
NG
MSA
NG
NG
PDA
05
0.5X10-5
158
15.8X10-5
EMB
98
9.8X10-5
MCA
NG
NG
SSA
NG
NG
MSA
NG
NG
NA
NA
NA
NA
10-4
10-4
10-4
10-4
25
06
0.6X10-5
06
0.6X10-5
01
0.1X10-5
NG
NG
SSA
NG
NG
MSA
02
0.2X10-5
PDA
10
1.0X10-5
03
0.6X10-5
EMB
NG
NG
MCA
NG
NG
SSA
NG
NG
MSA
NG
NG
PDA
02
0.2X10-5
78
7.8X10-5
EMB
03
0.3X10-5
MCA
NG
NG
SSA
NG
NG
MSA
NG
NG
PDA
NG
NG
PDA
ONIPANU (E)
NA
10-4
EMB
MCA
BARIGA (F)
SOMOLU (G)
NA
NA
10-4
10-4
10-4
LEGEND:
NA = Nutrient agar, EMB = Eosin methylene blue agar, MCA = Mackoncey agar, MSA = Manitol salt agar
SSA = Salmonella shigella agar, NG = No growth
26
TOTAL MICROBIAL LOAD COUNT(X10-5)
18
16
14
12
10
8
6
4
2
0
OYINGBO
IJORA
SABO
MUSHIN
ONIPANU
BARIGA
SHOMOLU
FIGURE 4.1: TOTAL MICROBIAL LOAD COUNT ON NUTRIENT AGAR FOR CHICKEN FROM DIFFERENT
LOCATIONS IN LAGOS STATE.
This figure above reveals the rate at which the various sample (frozen chicken) is contaminated
microbiological. The sample given some market in Lagos metropolis namely, Oyingbo, Ijora,
Sabo, Mushin, Bariga, Somolu and Onipanu. The total microbial load using an all purpose media
(nutrient Agar) was conducted across all the sample from the various locations. It was discovered
that the sample from Mushin yield the highest microbial load of 15.8×10-5 while The lowest count
was recorded at bargain with the load of 0.6×10-3
27
TOTAL MICROBIAL LOAD COUNT(X10-5)
12
10
8
6
4
2
0
OYINGBO
IJORA
SABO
MUSHIN
ONIPANU
BARIGA
SHOMOLU
FIGURE 4.2: TOTAL MICROBIAL LOAD COUNT ON EMB FOR CHICKEN FROM DIFFERENT LOCATIONS IN
LAGOS STATE.
Figure 4.2: The total enteric bacterials (coliform)load count in Emb was conducted with all across
all the sample from various locations. It show that mushin yielded the highest enteric bacteria with
loads of 9.8×10 while bariga is the lowest with the loads of 0.1×10
28
TOTAL MICROBIAL LOAD COUNT(X10-5)
1.2
1
0.8
0.6
0.4
0.2
0
OYINGBO
IJORA
SABO
MUSHIN
ONIPANU
BARIGA
SHOMOLU
FIGURE 4.3: TOTAL MICROBIAL LOAD COUNT ON PDA FOR CHICKEN FROM DIFFERENT LOCATIONS IN
LAGOS STATE.
Figure 4.3: As it represented in the bar chart indicate the fungi bacteria loads count in PDA was
conducted with various sample in different locations which shown that onipanu is the highest fungi
with loads 1×10 which somolu was recorded load of 0.1×10 which is the lowest.
29
Table 2 shows the morphological characteristics of the isolated organisms as a means of first stage identification using the following
parameters: shape, colour, size, edge, texture, surface, appearance and transparency. Many organisms isolated here, shows varying
morphological features among them selfies.
Table 4.2: Morphological characteristics of isolated bacteria
S/N
SAMPLE
MEDIA
COLOR
LOCATION
1
2
ONYNGBO
IJORA
SIZE
SHAPE
EDGE
(mm)
NA
SURFACE
TEXTURE
ELEVATION
TRANSPARENCY
APPEARANC
Yellow
5
Regular
Entire
Shiny
Soft
Raised
Opaque
White
12
Irregular
Undulate
Dry
Dry
Flat
Opaque
Cream
5
Regular
Entire
Shiny
Soft
Raised
Opaque
EMB
Purple
4
Regular
Entire
Shiny
Soft
Raised
Opaque
MCA
NG
NG
NG
NG
NG
NG
NG
NG
SSA
NG
NG
NG
NG
NG
NG
NG
NG
MSA
NG
NG
NG
NG
NG
NG
NG
NG
PDA
Black
18
Irregular
Undulate
Dry
Dry
Raised
Opaque
NA
Yellow
14
Regular
Entire
Shiny
Soft
Raised
Opaque
White
09
Irregular
Undulate
Dry
Dry
Flat
Opaque
Cream
05
Regular
Entire
Shiny
Soft
Raised
Opaque
Purple
2
Regular
Entire
Shiny
Soft
Raised
Opaque
EMB
30
3
4
SABO
MUSHIN
MCA
NG
NG
NG
NG
NG
NG
NG
NG
SSA
NG
NG
NG
NG
NG
NG
NG
NG
MSA
NG
NG
NG
NG
NG
NG
NG
NG
PDA
Black
18
Irregular
Undulate
Dry
Dry
Raised
Opaque
NA
Cream
4
Regular
Entire
Shiny
Soft
Raised
Opaque
Yellow
4
Regular
Entire
Shiny
Soft
Flat
Opaque
EMB
Purple
1
Regular
Entire
Shiny
Soft
Raised
Opaque
MCA
NG
NG
NG
NG
NG
NG
NG
NG
SSA
NG
NG
NG
NG
NG
NG
NG
NG
MSA
NG
NG
NG
NG
NG
NG
NG
NG
PDA
Green
10
Irregular
Undulate
Dry
Dry
Raised
Opaque
NA
Cream
3
Regular
Entire
Shiny
Soft
Raised
Opaque
Yellow
4
Regular
Entire
Shiny
Soft
Flat
Opaque
Purple
5
Regular
Entire
Shiny
Soft
Raised
Opaque
Green
3
Regular
Entire
Shiny
Soft
Raised
Opaque
EMB
metallic
sheen
MCA
NG
NG
NG
NG
NG
NG
NG
NG
SSA
NG
NG
NG
NG
NG
NG
NG
NG
31
5
6
7
ONIPANU
BARIGA
SOMOLU
MSA
NG
NG
NG
NG
NG
NG
NG
NG
PDA
Green
10
Irregular
Undulate
Dry
Dry
Raised
Opaque
NA
Cream
2
Regular
Entire
Shiny
Soft
Raised
Opaque
EMB
Purple
3
Regular
Entire
Shiny
Soft
Raised
Opaque
MCA
NG
NG
NG
NG
NG
NG
NG
NG
SSA
NG
NG
NG
NG
NG
NG
NG
NG
MSA
NG
NG
NG
NG
NG
NG
NG
NG
PDA
Green
10
Irregular
Undulate
Dry
Dry
Raised
Opaque
NA
White
8
Regular
Entire
Hard
dry
Flat
Opaque
EMB
Purple
3
Regular
Entire
Shiny
Soft
Raised
Opaque
MCA
NG
NG
NG
NG
NG
NG
NG
NG
SSA
NG
NG
NG
NG
NG
NG
NG
NG
MSA
NG
NG
NG
NG
NG
NG
NG
NG
PDA
Green
10
Irregular
Undulate
Dry
Dry
Raised
Opaque
NA
Yellow
2
Regular
Entire
Shiny
Soft
Flat
Opaque
cream
6
Irregular
Undulate
Dry
hard
Flat
Opaque
EMB
Purple
5
Regular
Entire
Shiny
Soft
Raised
Opaque
MCA
NG
NG
NG
NG
NG
NG
NG
NG
SSA
NG
NG
NG
NG
NG
NG
NG
NG
32
MSA
NG
NG
NG
NG
NG
NG
NG
NG
PDA
Green
10
Irregular
Undulate
Dry
Dry
Raised
Opaque
LEGEND:
NA = Nutrient agar
EMB = Eosin methylene blue agar
MCA = Mackoncey agar
MSA = Manitol salt agar
SSA = Salmonella shigella agar
NG = No growth
33
TABLE 4.3: SHOWING MORPHOLOGICAL CHARACTERISTICS AND IDENTIFICATION OF THE ISOLATED
FUNGI
This table revealing the morphological characteristics as a means of identification of the isolated fungi using the lactophenol cotton blue
staining techniques and the physical appearance of these fungi on culture plates. The colours of both the front and back view were
determined and recorded. The following fungi were suspected; Trichoderma spp, Aspergillus niger, Aspergillus fumigatus and
Aspergillus flavus.
34
ISOLATE
1
COLONIAL
MICROSCOPIC
MACROSCOPICAL
MACROSCOPICAL
SUSPECTED
MOPHOLOGY
VIEW
FRONT VIEW
BACK VIEW
FUNGI
(COLOUR)
(COLOUR)
Fluffy greenish
Presence of septate Greenish
surface with an
hyphae with
irregular shape
cornidiophores
Greenish-yellow
Trichoderma spp
Aspergillus niger
bearing spores
2
Black surface
Posses a very large Black
White with Yellow
surrounded with
globular spores
ring
a whitish
heads with a
background
septate hyphae
having a round
conidia
3
Greenish
Posseses a non-
Greenish white(off-
white(off-white)
septate hyphae
white)
Yellowish
Aspergillus
fumigatus
35
in dried and
powdry form
4
Greenish dry
Posseses a non-
texture
septate hyphae
Greenish
Yellowish
36
Aspergillus flavus
A
C
B
D
LEGEND:
A = Photomicrograph of Trichoderma sp
B = Photomicrograph of Aspergillus fumigatus
C = Photomicrograph of Aspergillus niger
D = Photomicrograph of Aspergillus flavus
37
CHAPTER FIVE
5.1
DISCUSSION
The microbial assessment of the quality and safety of food is important in human health. A high
bacteria colony in food sample simply suggests non-hygienic handing, poor storage, and
inadequate general hygiene during processing and/or poor quality raw materials (Newsome, 1988).
The present study evaluated the bacteriological quality of frozen chicken at retail points in seven
different markets in Lagos state namely; Onyingbo, Ijora, Sabo, Mushin, Onipanu, Bariga and
Somolu markets respectively. A total of eight (8) frozen chicken samples from the above listed
locations were analyzed microbiologically to ascertain their level of microbial contamination and
possibly presence of virulent pathogens. Table 1 shows the morphological characteristic as a
means of identification by determining physical appearances of the isolate such as color, shape,
edges (margin), elevation, and consistency. Table 2 reveals the level of microbial load in the
various samples across all the locations. However the result shows that all the Nutrient agar and
Eosin methylene blue agar media yielded growth of microorganism. The overall microbial
population across all the samples from the various locations reveals that Mushin sample yielded
the highest microbial load with 15.8x105 while the least microbial population was recorded as
0.3x105 from Bariga.
Table 3 reveals the biochemical tests for identification of the bacteria. From the result, more gram
negative organisms such as Escherichia coli, Enterobacter spp and Citrobacter spp were isolated
than the gram positive bacteria such as Staphylococcus aureus and bacillus spp. The result
obtained in this research work is in conformity with Oritsetimeyin, 2019. The presence of Gram
negative bacteria (lactose fermenters), is an indication of contamination by coliforms. The
presence of Escherichia coli samples indicates the possibility of a microbial hazard and fecal
38
contamination. Coliforms are considered as normal flora of the intestinal tract of humans and
animals. They have been used as indictor organisms for bacteriological quality of food and water.
Escherichia coli is one of the most important food borne bacteria pathogens in ready to eat food..
The presence of E. coli in the samples suggested a very poor level of sanitation either during or
after storage. Bacillus which is known to be among the isolated bacteria causes toxin medicated
disease rather than an infection. Bacillus spp, is a normal inhabitant of the soil and a poisoning
organism associated with animals. This is however, because of the survival advantage which the
spores have in air and in other harsh condition. Bacillus cereus isolated from these samples could
come from soil dust since large number of Bacillus spp. occurs in the soil (Atlass, 1997). The
pathogenicity test carried out revealed that all the isolated bacteria were non virulent as thus could
not cause more treat to life.
5.2
CONCLUSION
Frozen chicken is one of the meats with high consumption rate and thus should be given much
attention by the stipulated regulatory body in Lagos states sequel to the result obtained. A good
personal and environmental hygiene should be put in place in other to reduce the high level of
microbial load recorded in some of the location.
39
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