CHAPTER ONE

INTRODUCTION

1.1 Background to the Study

Meat is consumed by many people as an important source of protein and other nutrients (Ekli et

al., 2020). It has been estimated that 62 billion chickens, 1.5 billion pigs, 545 million sheep, 444

million goats, and 301 million cattle were slaughtered for meat consumption worldwide in 2014.

Furthermore, pork is the most consumed meat (average consumption of 16 kg per year in 2013),

followed by poultry (15 kg), beef/buffalo (9 kg), and mutton and chevon (2 kg) (Ritchie and

Roser, 2019). Meat consumption is known to be highest across high-income countries and lowest

in low-income countries (Ritchie and Roser, 2019). Speedy reported that the United States of

America is the leading consumer of meat in the world with 124 kg/capita/year. Africa and South

Asia are the least consumers of meat with a consumption of between 3 and 5 k/capita/year

(Speedy, 2019).

Most meats have high water content corresponding to a water activity of approximately 0.99

which is suitable for microbial growth (Rao V. A., Thulasi G., Ruban S. W. (2009)). Microbial

growth can lead to food spoilage and foodborne infections in humans, resulting in economic and

health losses (Rao et al., 2018). Some strains of Escherichia coli (E. coli) are among the

pathogens that have been associated with foodborne infections in humans. Some of the

foodborne infections in humans have also occurred as a result of the consumption of

contaminated meats. For instance, the Centers for Disease Control and Prevention reported an

outbreak of E. coli infections linked to the consumption of ground beef which resulted in 29

hospitalizations and 0 deaths. A more serious E. coli outbreak linked to the consumption of

ground beef occurred in 2018 which led to 1 death and 6 hospitalizations (Komba et al., 2012).

1
In the European Union, 6,073 confirmed cases of Shiga toxin-producing E. coli (STEC)

infections were reported in 2017. There were 20 deaths (case fatality of 0.5%), and the

contribution of STEC from animal sources was found (Komba et al., 2012).

E. coli is a common bacterial contaminant of meat, and most strains of E. coli are commensal

enteric organisms. Their presence in food products indicates direct or indirect fecal

contamination that most likely occurs due to deficits in hygiene during product preparation.

Although most commensal E. coli strains are hargmess, there are many strains that are harmful to

people. Furthermore, commensal E. coli are thought to serve as reservoirs for antimicrobial

resistance and associated antimicrobial resistance genes that can be shared with pathogens.

Antibiotic resistance in pathogens can complicate treatment of infectious disease, leading to

prolonged hospitalization, treatment failure, and death. In Tanzania, the occurrence of

antimicrobial-resistant (AMR) infections has been reported (Centers for Disease Control

Prevention, 2018)

Although most foodborne infections are self-limited, antimicrobials are used when necessary.

The use of antimicrobials has resulted in the development of resistant pathogens including E.

coli, which is a threat to public health. To accurately study the involvement of microorganisms in

foodborne infections, robust tools/methods that will ensure effective isolation, phenotypic,

and/or genetic characterization are required. Research has demonstrated that meat samples in

Africa are contaminated by E. coli (European Food Safety Authority, 2017).

The burden of foodborne diseases in Africa has become a major public health concern due to

Africa’s numerous infrastructural challenges. Enterohaemorrhagic Escherichia coli O157:H7 is

one of the most important foodborne pathogens. It often causes significant mortality among the

human population. Typical illnesses as a result of this pathogen can be life threatening.

2
Susceptible individuals show a range of symptoms, including haemorrhagic colitis and other

complications, such as haemolytic uremic syndrome and thrombotic thrombocytopenic purpura.

Domestic ruminants, including cows, are the natural reservoirs of E. coli O157:H7, and they play

a significant role in the epidemiology of human infections (Adzitey, 2015).

E. coli O157:H7 is carried in the intestinal tract, excreted in animal waste and may be transferred

to carcasses during slaughter and evisceration. Human infection is via the faecal–oral route. This

often results from ingestion of contaminated and improperly cooked meat and meat products.

Nigeria ranks first in the health burden of zoonotic diseases in Africa. It was categorized into a

sub-region that experiences the highest burden of foodborne disease globally, with E. coli O157

identified as one of the leading causes of foodborne disability adjusted life years (Adzitey, 2015).

The use of antibiotics for the treatment of E. coli O157:H7 infections has been very

controversial. A study had reported that antibiotics may worsen the illness and increase the risk

of haemolytic uremic syndrome, due to increased toxin production. In contrast, early

administration of some antibiotics is reported to be effective. However, the frequent misuse of

antibiotics as a growth-promoting supplement in animal feed creates a huge pressure for the

selection of antibiotic resistant strains among pathogenic bacteria, and contributes significantly

to the emergence and spread of antibiotic resistance (Adzitey, 2015).

The lack of surveillance systems for E. coli O157:H7, poor and sub-standard hygienic

conditions, unhygienic slaughter practices in abattoirs and the common traditional practices of

raw meat consumption by certain tribes in Nigeria, constitute the major factors that predispose

the local tribes to high risk of exposure and may possibly spread the pathogen to other Nigerians.

In addition, there is paucity of information regarding the epidemiology of E. coli O157:H7 in

Nigeria.

3
To bridge this knowledge gap, this study was designed to assess the level of meat contamination

with E. coli O157:H7 in beef at a commercial slaughterhouse in Toru-orua Bayelsa State,

Nigeria; test the susceptibility of the isolates to a panel of antibiotic agents; and assess the

hygienic practices at the abattoir during the slaughtering process (Adzitey, 2015).

Meat as Food

Meat is flesh of an animal that is eaten as food (Lawrie and Ledward, 2006). Meat is also defined

by the Codex Alimentarius as all parts of an animal that are intended for or have been judged as

safe and suitable for human consumption from the nutritional point of view (CAC, 2005). The

primary unit of meat is called carcass. It represents the ideal meat after removal of the head, hide,

intestine and blood (Rao et al., 2009). Most often meat refers to the skeletal muscle , associated

fat and other tissues, but it may also describe other edible tissues such as offals (i.e. meat other

than meat flesh, including brain, heart, kidney, liver, pancreas, spleen, thymus, and tongue)

(Lawrie and Ledward, 2006). The advent of civilization allowed the domestication of animals

such as chickens, sheep, pigs and cattle, and eventually their use in meat production on an

industrial scale (Robert 2000). Meat is produced by killing an animal and cutting flesh out of it.

These procedures are called slaughter and butchery respectively. There is ongoing research into

producing meat in -vitro that is, outside of animals (Twum, 2016).

Meat can be broadly classified as "red" or "white" depending on the concentration of myoglobin

in the muscle fiber. When myoglobin is exposed to oxygen, reddish oxymyoglobin develops,

making myoglobin-rich meat appear red. The redness of meat depends on species, animal age,

and fiber type. Red meat contains more narrow muscle fibers that tend to operate over long

periods without rest, while white meat contains more broad fibers that tend to work in short fast

4
bursts. The meat of adult mammals such as cows, sheep, goats, and horses is generally

considered red, while chicken and turkey meat is generally considered white (Lawrie and

Ledward, 2006).

The nutritional compositions of red meat changes depending on breed, feeding, season and meat

cut. However lean red meat shows consistency in high protein content, essential vitamins and

minerals, relatively low-fat content and moderate in cholesterol (Williams, 2007). Meat is a

nutritious food as the protein of the meat required by man and also is an excellent source of iron,

phosphorus, potassium, and sodium (Rao et al., 2009), meat is also an important source of the B

vitamins, particularly B1 (thiamine), niacin (nicotinic acid), B2 (riboflavin), B6 and B12

(cyanocobalamin) and vitamin A (retinol). It is a major source of iron, copper, zinc, and some

selenium (Warriss, 2010). Butcher meat is a valuable part of the human diet because (a) it is the

most concentrated and is a good source of first-class protein that is, it contains those amino acids

which are essential for human life; (b) it stimulates metabolism due to its high protein content,

that is to say, it assists the body in the production of heat and energy; (c) it is satisfying, for the

presence of fat in the diet delays emptying of the stomach (Eroclini et al., 2006).

The term meat quality is used to describe a range of attributes of meat. Many factors determine

the quality of meat. It includes requirements of food safety and animal welfare. It also includes

the sensory appeal of meat such as palatability (visual appearance, smell, firmness, juiciness,

tenderness, and flavor) and perceived healthiness, especially in relation to the amount and type of

fat and other fatty components (Aberle, E.D., Forrest, J.C., & Gerrard, D. & Mills, E.W. (2001).

Quality of meat describes how attractive the meat is to consumers. Meat must look good to

consumers before satisfying their palate when they decide to buy it. The expectations of the

consumer in terms of aroma, tenderness, juiciness, flavor, color, wholesomeness and nutrition

5
must be met once the meat is bought, cooked, and served, (Aberle et al., 2001). Flavor is

interwoven with an aroma to bring out the sensation the consumer has during eating. Flavor and

aroma are perceptions and depend on the ability to smell through the nose and on the sensations

of salty, sweet, sour and bitter on the tongue. Meat flavor is affected by the type of species, diet,

cooking method and method of preservation (e.g. smoked or cured) (FAO, 2003). The source of

flavor in meat is the fat, the different flavors among a different kind of meat (beef, pork, chicken,

turkey, and mutton) come from fatty components. Fat acts as one of the precursors of flavor by

combining with amino acids from proteins and 3 other components when heated. The aroma and

juiciness of meat products can be improved using spices and cooking method. The tenderness

depends on textural characteristics, the composition of meat, breeds, sex, and many other factors.

Tenderness of meat is also based on ease of chewing, which is contributed by the fibrous nature

of muscle (Gerrard and Grant, 2003).

1.2 Statement of Problem

Pathogenic organisms are organisms that are referred to as specific health hazards associated

with gastrointestinal disturbance counting foxiness. The victim suffering from abdominal pain

and diarrhea with more vomiting thus diarrhea usually manifest the illness which when

untreated, in some cases where it does not result to death, it leads to unnecessary expenses in

seeking for medical advice. This has now made interest on how we can improve on our food

hygiene to avoid contamination.

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1.3 Justification

According to the research work, Pathogenic organisms collected from raw meat gotten from

Kpansia market are highly risky and not fit for human consumption and pose a threat to

human health. As a result in some cases where it does not result to death, it leads to

unnecessary expenses in seeking for medical advice on how to treat such infection.

1.3 Aims

The aim of this project is to carry out the microbial examination of raw meat obtained at

Toru-Orua abattoir

1.4 Objective

The objectives of this study are:-

 To isolate microorganisms from raw meat sold in Toru-Orua abattoir

 To study the prevalence of E.coli from raw meat sample

1.5 Significance of the Study

The significance of this work is to enable those involved in cow slaughtering to understand the

importance of keeping abattoir premises clean and tidy. This can be achieved by:

 Keeping abattoir clean at all times most especially after killing of animal

 Ensuring that during preparation, food is in the danger zone for as short time as

possible.

 Using suitable preservatives such as salt.

 Using various suitable packaging methods.

7
 CHAPTERS TWO

LITERATURE REVIEW

2.1 Factors Affecting the Growth of Microorganisms in meat

A better understanding of the various influences can help in preventing the microbial growth and

hence, avert spoilage of the meat and meat products. The factors affecting microbial growth in

livestock products can be broadly divided into two categories namely intrinsic factor and

extrinsic factor.

2.1.1 Intrinsic Factors

Intrinsic factors are those factors that are characteristic of the product itself.

2.1.1.1 Moisture content and water activity

Microorganisms need water in an available form to grow in food products. The control of the

moisture content in foods is one of the oldest exploited preservation strategies. Food

microbiologists generally describe the water requirements of microorganisms in terms of the

water activity (aw) of the food or environment. Water activity is defined as the ratio of water

vapor pressure of the food substrate to the vapor pressure of pure water at the same temperature

(Jay JM 2000):

Microbes require at least 13 per cent free water for their growth and it is best expressed in terms

of available water or water activity (aw). The aw of pure water is 1.0 and many bacteria, yeasts

and moulds require 0.91, 0.88 and 0.80 aw, respectively. Foods preserved with salt or sugar

concentration do not support growth of most microbes. For example salt concentration of 5-15

per cent inhibits growth of bacteria whereas many moulds and some yeasts can tolerate more

8
than 15 per cent. Sugar concentration of 65 per cent and above is required to inhibit mould

growth whereas 50 per cent do not allow bacteria and most yeasts (Bhat et al., 2012).

2.1.1.2 Nutrient content

Like other living organisms, microorganisms need nutrients for growth and maintenance. These

nutrients are used as sources of energy, carbon, nitrogen, minerals, vitamins, and other growth

factors. The ability of a food item to support microbial growth depends on its content of these

nutrients. The major nutrients used by microorganisms as sources of energy, carbon, and

nitrogen are the carbohydrates, proteins, and lipids.Foods rich in carbohydrate can be spoiled by

carbohydrate-fermenting microorganisms, particularly by yeasts and molds. Bacterial species of

the genera Bacillus, Clostridium, Aeromonas, Pseudomonas, Leuconostoc, and Enterobacter are

saccharolytic and can also attack carbohydrates (Banwart, 2004; Ray, 2004)

Most microorganisms need one or another vitamin as growth factors, normally in small amounts,

and almost all natural human foods contain enough of such vitamins to support microbial

growth. In general, Gram-positive bacteria are more fastidious in their needs for vitamins, while

Gram-negative bacteria and molds can synthesize most or all of their needs from these growth

factors (Jay, 2000). Foods contain7 the nutrients required for the growth of microorganisms in

varying quantities. Milk contains all nutrients in sufficient amounts that support the growth of

almost all types of microorganisms. Meats are rich in proteins, lipids, minerals, and vitamins, but

poor in carbohydrates, while foods of plant origin are generally rich in carbohydrate, but

relatively poor in proteins, minerals, and some vitamins (Ray, 2004)

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2.1.2 Extrinsic factors

Extrinsic factors are those factors that refer to the environment surrounding the food.

2.1.2.1 Temperature

The growth of organisms is caused by the environmental temperatures. Few organisms are able

to grow at certain temperatures. Psychrophilic organisms grow best at about 20°C but also down

to -10°C in unfrozen media. Mesophilic organisms grow between 25°C and 40°C with an

optimum growth temperature close to 37°C none of the mesophilic bacteria are able to grow

below 5°C or above 45°C pathogenic bacteria belong to this group. Thermophilic organisms

grow at temperatures above 45°C, their optimum growth temperatures is between 50°C and 70°C

mostly this bacteria are found in food industry especially in processed foods. (Decaille Donna

2021).

2.2 Common Microbial Present in Meat and Meat Products

Microorganisms of relevance with regard to meat hygiene include helminths, moulds, bacteria

and viruses. Within these groups, bacteria play the most important role. Parasites are of

insignificant value in meat which has passed meat inspection, or where efficient internal parasite

control programmes or measure are in place. The most frequently identified bacterial pathogen

associated with consumption of beef products are Salmonella spp, Compylobacter spp,

Staphylococcus aureus, Escherichia coli, Listeria 6 monocytogenes, Clostridium perfringens,

Yersinia enterocolitica, Bacillus cereus and Vibrio parahaemolyticus (Biswas, A. J., Kondaiah,

N., Anjaneyulu, A. S. R. & Mandal, P. K. 2011)). Compylobacter spp, Salmonella spp and

Escherichia coli are often present in fresh meat and poultry (Zhao, C., Ge, B., DeVillena, J.,

10
Sudler, R., Yeh, E., White, D. G., Wagner, D. and Meng, J.(2001).). (Ali, N. H., Farooqui, A.,

Khan, A., Khan, A.Y. and Kazmi, S.U. 2010) reported the foodborne pathogens isolated from

meat samples in retail meat shops. They included Escherichia coli O157:H7, Listeria spp,

Salmonella enteritidis and Shigella species while in meat handling equipments in retail shops

were Staphylococcus and Shigella spp. Isolated Staphylococcus aureus, Bacillus cereus,

Clostridium perfringens and Escherichia coli in beef samples from butchers. Moreover, the

faecal coliforms such as Escherichia coli are generally considered as indisputable indicators of

faecal contamination from warm blooded animals (Yousuf, A. H. M., Ahmed, M. K., Yeasmin,

S., Ahsan, N., Rahman, M. M. and Islam, M. M. (2008).

2.3 The Effects of Bacteria in Meat and Meat Products

Food animals are useful as they supply quality protein and revenues to man, but on the other

hand they serve as vehicles of disease pathogens. Raw meat remains an important and probably

the major source of human food borne infection with pathogenic bacteria. In spite of decades of

effort to control them, it has been difficult to obtain food animals free of pathogenic bacteria

(Wilfred and Fairoze, 2011). The effects that microbial contaminants cause on meat include

spoilage of the meat, food poisoning and condemnation of carcasses which results into reduction

of income to farmers as well as meat sellers. Consumers and meat handlers may acquire bacterial

diseases such as Anthrax, Q-fever, Campylobacteriosis, Ornithosis, Botulism, Staphylococcus

food poisoning, Salmonellosis, Brucellosis, Erysipelas, Streptococcosis, Tetanus, Yersiniosis,

Clostridiosis, Listeriosis, Glanders, Leptospirosis and Tuberculosis due to poor handling of food

animals and meat (Adeyemo, 2002).

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2.4 Source of Beef Contamination

Unless the animals are infected, the meat of freshly slaughtered animals are generally sterile. The

presence of microorganisms on post slaughtered carcasses is due to contamination occurring

immediately before, during and after slaughter. The microbial contaminations of carcasses occur

mainly during processing and manipulation during skinning, evisceration, processing at abattoir

and retailers establishments (Gill, 1998). The main sources of meat contamination include;

animal/carcasses source, on farm factors, transport factors, abattoir and butchers facilities,

parasites and wild animals, meat van, abattoir and retail meat outlet workers.

2.4.1 Animal/carcasses source

Faecal matter is a major source of contamination and can reach carcasses through direct

deposition as well as by indirect contact through contaminated carcasses, equipments, workers,

installations and air (Borch and Arinder, 2002). Faeces as well as soil adhering to animals are

carried into abattoir on hair, hides, hooves and tail of animals. Contact between carcasses and

hides allow a mixture of microorganisms to be introduced on the carcasses. These contaminating

microorganisms are derived from the animal’s pre slaughter environment that may be of faecal,

soil, water or feed origin (Bell, 1997). Infected body fluid such as urine, milk, blood, mucus,

rumen fluid, intestinal fluid and fluid from excised abscess can be another source of carcasses

contamination (Galland, 1997).

2.4.2 On farm factors

Body condition may affect the pathogens load. Weak animals lie down more often than healthy

ones, thereby increasing the likelihood of contaminating hides. Contacts between animals at

12
auction barns may increase the pathogen load (Galland, 1997). The exterior of the animals

harbours large number and different types of microorganisms from soil, water, feed, manure as

well as its natural flora.

2.4.3 Transportation of slaughter animals

The transport factors such as the type and cleanliness of transport facility, distance travelled and

duration of journey, harshness of ride, overpopulation of animals in the conveyance and

frequency of stops, may affect and contribute to pathogen load (Galland, 1997).

2.4.4 Abattoir and butchers facilities

The abattoir and beef retail outlet environments play important roles in contamination of meat.

Site selection and availability of good quality portable water are important factors to consider

when selecting site for constructing abattoir or retail meat outlets since it affects the quality of

meat. Meat contamination in abattoirs and retail meat outlets result from the use of contaminated

water, unhygienic practices like poor handling, use of contaminated tables to display meat

intended for sale and the use of contaminated knives and other equipments in cutting operations

(Fasanmi, G. O., Olukole, S. G. & Kehinde, O. O. 2010).

The length of time animals are held at the abattoir before slaughter can affect the pathogen load

by increasing the probability of exposure and infections. Sanitation of walk ways, pen floor,

railings, feed and water affect the pathogen load (Galland, 1997). Dirt, soil, body discharges and

excreta from animals in holding pens or lairages are primary sources of contamination of

carcasses in the later stages of the operation. This happens irrespective of whether or not the

animals are fit and have passed ante mortem inspection. Adzitey, F., Teye, G. A., Kutah, W. N.

& Adday, S.2011a) reported the possible sources of contaminations arising from the cutting

13
knives, intestinal contents, chopping boards, hides, meat handlers, containers, vehicle for

transporting carcasses and the meat selling environment. It has been reported by Ali et al. (2010)

that knives, wooden boards and weighing scales from retail shops are sources of bacterial

contamination particularly Staphylococcus aureus and Shigella species. (Akinro, A. O.,

Ologunagba, I. B. & Yahaya, O. 2009).) reported that with inadequate slaughtering and disposal

facilities, the abattoir becomes a source of infection and pollution, attracting domestic and wild

carnivores, rodents and flies, which are vectors of diseases. Refrigerator or freezers are essential

storage facilities used to prevent spoilage of meat following prolonged storage at room

temperature and hence keep meat safe for long period of time.

2.4.5 Parasites and wild animals

With inadequate slaughtering and disposal facilities attracting flies, domestic animals, wild

carnivores and rodents, abattoir/slaughter houses become among the important sources of

microbial contamination (Adeyemo, 2002).\

2.4.6 Meat van

The vehicles used to transport meat from abattoir to retail meat outlets may act as sources of

contamination since often lack regular cleanliness and are not well covered leading to

contamination by dusts, insects and flies. Sulley, (2006) reported contamination of meat resulting

from other means of transport such as motor-bikes and bicycles due to insufficient vans and

trucks. On the other hand, the few transport available were not properly cleaned and thus

contained high microbial loads (Sulley, 2006).

14
2.4.7 Abattoir and retail meat outlet workers

The hygienic condition of the abattoir and retail meat outlet workers has potential to contribute

contamination in beef before and after processing. Adetunde, L. A., Glover, R. L. K., Oliver, A.

W. O. and Samuel, T. (2011) reported that unclean slaughter men’s hands, butcher arms,

clothing and equipment used in carcass dressing process accounted for the microbial

contamination and also the study of Jeffery, (2003) revealed that the worker hands and their

equipments were among the main sources of meat contamination.

15
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Sample Location`

Raw meet in this study, where bought from meat seller withing Toru-orua Bayelsa state. A total

of two samples were collected one sample each were collected from Abattoir A and Abattoir B is

located at, opposite the University back gate and T-junction inside the Toru-orua community

respectively.

3.2 Sample Collection

Raw meats used in this study were bought from meat sellers within Toru-orua community,

Bayelsa state. A total of two samples were collected, one sample was collected from Abattoir A

and one sample from Abattoir B and were put inside a sterile universal bottle, and taken to the

microbiology laboratory for analysis.

3.3 Sterilization of Materials

All the glass wares used for the experiment were sterilized using the laboratory hot air oven at a

temperature of 160℃ for 1 hour while media was autoclaved at a temperature of 121 ℃ for 15

minutes at 15psi. Wire loop was sterilized over burning flame and allowed till its red-hot, while

glass spreader was sterilized by dipping into 70 % ethanol and passing over Bunsen flame.

3.4 Media

The media used in this study; Nutrient agar, Simmon citrate agar, peptone water and Triple sugar

ion agar were prepared according to manufacturer’s instructions and sterilized using the

16
autoclave at a temperature of 121℃ at 15psi for 15minutes and were allowed to cool to a

temperature of 45℃ and about 20 millilitres was poured into sterile petri-dishes. The plates were

allowed to cool and set for inoculation.

3.5 Serial Dilution

The serial dilution method as described by Falegan et al. (2017) was adopted. Ten grams (10g)

each of the meat samples were weighed and mashed in a laboratory type mortar and pestle into a

paste. Thereafter, each mashed sample was added into different clean sterile beakers containing

ninety millilitres (90gm) of sterile water (stock). The beakers were shaken at intervals for 30

minutes. Nine (9) millilitres of sterile water was added into six (6) sets of test tubes. Thereafter a

six-fold serial dilution was done. One millilitre from each of the stock was transferred to the first

test tubes containing nine millilitres of sterile water using sterile pasteur pipettes for viable

counting. The test tubes were shaken and one millilitre was transferred to the second test tubes.

This procedure was continued to the last test tubes and one millilitre was discarded from each of

the last test tubes.

3.6 Inoculation of Samples

The spread plate technique as described by Cheesbrough (2010) was used in the inoculation of

the plates. 0.1 millilitre aliquot of the serially diluted samples was pipette from each of the test

tubes labelled 105 & 106 for bacteria and 10² & 104 for fungi and was dropped onto the different

media in the plates. A sterile bent glass rod was used to spread the aliquot evenly on the media.

The plates were labeled accordingly. The inoculated plates were inverted and incubated in the

incubator at a temperature of 37℃ for 24 hours for bacteria, and 72 hours for fungi.

17
3.7 Microbial Plate Count

After the incubation of the different plates, the different colonies formed on the media were

counted.

3.8 Purification and Preservation of Isolates

After the various colony counts, bacterial isolates were picked with a wire loop based on their

morphological appearances. The picked colonies were sub-cultured onto freshly prepared

nutrient agar plates to obtain pure cultures. They were further incubated for 24hrs at 37 0C. After

incubation pure cultures were stored in McCartney bottle in a refrigerator at 40 0C. Fungal

isolates were sub-cultured onto freshly prepared Sabouraud dextrose medium and also preserved.

3.9 Identification of Bacterial Isolates

3.9.1 Gram Staining Techniques

A smear of each of the bacterial isolates was made and fixed by air drying. The smears were then

covered with crystal violet stain for 60 seconds and rapidly washed off with water thereafter. The

smears were then covered with Lugol’s iodine for 60 seconds and washed off with water. The

smears were decolorized with acetone alcohol and washed off after 10 seconds. The smears were

finally flooded with safranin for 2minutes and washed- off with clean water. The back of the

slides were then wiped and placed in a draining rack for the smear to dry before they were

viewed with x 10 oil immersion objective lens (Cheesbrough, 2010). Gram positive bacteria gave

purple coloration while gram negative bacteria gave pinkish coloration.

3.9.2 Catalase Test

Three millilitres (3gm) of hydrogen peroxide was poured in a test tube. A colony of test

organism was taken with sterile wooden or glass rod and immersed into hydrogen peroxide

18
solution. Generation of bubbles indicated oxygen production. If bubbles were produced, the

organism was catalase-positive. However, if bubbles were not produced, the organism was

catalase-negative. (Cheesbrough 2010) .

3.9.3. Oxidase Test

A piece of filter paper was placed in Petri-dish and 3 drops of freshly prepared oxidase reagent

were added. Using a sterile glass rod, a colony of test organisms was removed from a culture

plate and smeared on the filter paper. Oxidase-positive organisms gave blue color within 5-10

seconds, and in oxidase-negative organisms, color did not change. (Cheesbrough 2010).

3.9.4. Citrate Test

A bacterial colony was inoculated in Simmons citrate agar and incubated at 35 to 37 ℃ for 18

to 24 h. Thereafter, development of blue color was observed. Citrate positive showed that growth

was visible on the slant surface and the medium became an intense blue while Citrate negative

showed trace or no growth was visible and no color change occurred. (Cheesbrough 2010).

3.9.5 Indole Test

Test bacterial colony were inoculated in peptone water and incubated at 37 ℃ for 24-28 h.

Thereafter, 0.5 gm of Kovac’s reagent was added. Positive test showed pink colored ring was

observed after addition of reagent. Negative test showed no color change after reagent addition.

(Cheesbrough 2010) .

3.9.6 Sugar Fermentation Test

Using stab inoculation, each colony of the different test organisms was inoculated onto sterile

19
agar slopes of triple sugar iron agar. The inoculated, agar slopes were incubated at 37 °C for 24

hours after this. The different colors of the slopes and butts, in addition to the presence of gas

production and hydrogen sulphide (H2S) blackening, were an indication of the type of bacteria

present. (Cheesbrough 2010) .

CHAPTER FOUR

20
 RESULTS AND DISCUSSION

4.1 Results

The result below shows the microbial load of the meat sample at the various abattoir represented

in Table 1. The total viable bacteria count ranges from 24.6×10-3. to 3.7×10-4 cfu/gm for abattoir

A and 6.1×10-3 to 3.8×10-4 for abattoir B, of which abattoir A have more growth than abattoir B.

Coliform count was detected with a range of 2×10-3 to 1.1×10-4 cfu/gm for abattoir A, and 2×10-4

to 3×10-5 for abattoir B. Table 2 shows the bacteria isolate of the various sample plate, the total

of three bacteria were isolated from the various plate sample from the different abattoir and they

include Yersinia, Escherichia coli and Salmonella spp are the result for the identification of

bacterial isolates. while Table 3 shows the result for the identification of the fungal isolates. The

fungal isolated from Abattoir A and Abattoir B are all Aspergillus spp.

Table 1: Total Microbial Load Of The Various Plate Sample

21
 Sample codes TVC(cfu/gm) TCC(cfu/gm) TFC(cfu/gm)

Abattoir A1 24.6×10-3 2×10-3 1×10-1

Abattoir A2 3.7×10-4 1.1×10-4 2×10-3

Abattoir B1 6.1×10-3 2×10-4 1×10-1

Abattoir B2 3.8×10-4 3×10-5 4×10-3

TVC = Total viable count

TCC = Total coliform count

TFC = Total fungal count

Table 2: Biochemical Characteristics of Bacterial Isolates

22
sample TSI TEST Probable

G L S G H2s microorganism

Morphology

Gram stain
Catalase

citrate
Deep pink, Rose - - + + - - - + - - Yersinia

pink, shiny, rough,

Abattoir
mucoid, flat, opaque
A&

Abattoir

Abattoir Yellowish, opaque, - + + - + + - + - - Escherichia coli

A & round, irregular,

Abattoir rough, mucoid, flat,

B raised

Abattoir Milkfish, - - + + + - - + + - Salmonella spp.

A & transparent, round,

Abattoir irregular, rough,

B mucoid, flat, raised

+ = positive, - = negative, G= glucose, L= Lactose, S= Sucrose, G= Gas production, H2S=

Hydrogen Sulphide production.

23
 AA 10-5 AB 10-5

AA 10-6 AB 10-6

AB 10-6 AA 10-5

AB 10-5 AA 10-6

AA- Abattoir A (opposite university back gate) : dilution factor 10-5 and 10-6

AB- Abattoir B (T-Junction inside Toru-Orua Community) : dilution factor 10-5 and 10-6

24
 2:
Salmonella spp
1:
Salmonella spp 3:
E. coli

5:
Yersinia 4:
Yersinia

Subculture from the above plates

25
Table 3: Identification of Fungal Isolates

SAMPLE CODE MACROSCOPIC MICROSCOPIC POSSIBLE FUNGI

APPEARANCE APPEARANCE

Abattoir A Dark brown to white Septate Hyphae with Aspergillus spp

surface, Raised, Opaque conidia bearing sterigmata

Abattoir A Dark brown to milkfish Septate Hyphae with Aspergillus spp

surface, Raised, Opaque conidia bearing sterigmata

Abattoir B White cottony colony, Conidial with visible Aspergillus spp

Raised, rhizod Hyphae

transparent

Abattoir B Green to white, raised, Smooth conidial with Aspergillus spp

opaque visible hyphae

Green to white, irregular ,

unbonated,

Milkish, rhizoid, raised

26
4.2 Discussion

This study was conducted to determine the microbial load and possible pathogens present in raw

meat obtained at abattoir A and B located in Toru-orua. community.

The result below shows the microbial load of the meat sample at the various abattoir represented

in Table 1. The total viable bacteria count ranges from 24.6×10-3. to 3.7×10-4 cfu/gm for abattoir

A and 6.1×10-3 to 3.8×10-4 for abattoir B, of which abattoir A have more growth than abattoir B.

Coliform count was detected with a range of 2×10 -3 to 1.1×10-4 cfu/gm for abattoir A, and 2×10-4

to 3×10-5 for abattoir B. Table 2 shows the bacteria isolate of the various sample plate, the total

of three bacteria were isolated from the various plate sample from the different abattoir and they

include Yersinia, Escherichia coli and Salmonella spp.

Table 3 shows the result for the identification of the fungal isolates. The fungal isolated from

Abattoir A and Abattoir B are all Aspergillus spp.

27
 CHAPTER FIVE

CONCLUSION AND RECOMMENDATION

5.1 Conclusion

The present study reveals the fact that raw meat from retail outlets is contaminated with the high

incidence of bacterial pathogens and low incidence of fungal pathogens. The antibiotic resistance

pattern of the bacterial isolates shows the high incidence of multi-drug resistant bacterial

contaminants in meat. This states the role of raw food as a reservoir of antibiotic resistant

bacteria which can be transferred to humans thereby causing gastrointestinal disorders and food

borne illness which can be life threatening. It is imperative that basic hygienic practices be

incorporated in abattoirs and retail meat outlets to ensure food safety. Training should be given

to meat handlers and butchers regarding food safety practices and proper inspection procedures

should be strictly adhered to minimise the contamination of raw meat and meat products sold in

market places.

5.3 Recommendations

Based on the findings of this research work, the researcher recommended the followings:

1. Proper cleaning mechanisms system should be deployed in the Abattoirs.

2. To protect the health of individuals, proper control measures has to be taken to prevent

the abattoir which facilitate the growth and proliferation of pathogenic bacteria in

abattoirs

3. Disinfection of tables, knifes and other equipment use in the abattoir should be performed

routinely

4. Finally, meat gotten from the abattoir should be washed and prepared properly before

consumption.

28
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