MICROBIAL ASSESSMENT OF FRESHLY CAUGHT FISH IN

OTAMIRI RIVER, OWERRI, IMO STATE

BY

CHUKWUNAGOROM JOY IFUNANYA

18H/0013/FIT

A PROJECT SUBMITTED TO THE DEPARTMENTOF FISHERIES

TECHNOLOGY,
SCHOOL OF AGRICULTURE AND AGRICULTURAL
TECHNOLOGY
FEDERAL POLYTECHNIC NEKEDE OWERRI, IMO STATE.

IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE

AWARD OF HIGHER NATIONAL DIPLOMA (HND) IN
FISHERIES TECHNOLOGY

SEPTEMBER, 2021
i
 CERTIFICATION
This is to certify that this project work on Microbial Assessment of freshly Caught
fish in Otamiri River, Imo State was carried out by CHUKWUNAGOROM JOY
IFUNANYA with the registration number 18H/0013/FIT, has been accepted and
approved by the Department of Fisheries Technology

……………………........ …………………………..
Mr. Nwaka S.U Date
(Project Supervisor)

………………………… …………………………..
Dr. Nwachukwu V.N Date
(Head of Department)

………………………. ………………………….
Dr. N. A. Jamabo
External Examiner Date
 DEDICATION
This project is dedicated to the Almighty God for his protection and guidance
during this work.
 ACKNOWLEDGEMENT
With utmost gratitude, I appreciate the mercies, protection, guidance, favor and
goodness of God to me.I am immensely grateful for the immeasurable support,
encouragement and advice of my loving husband and my family. I wish to express
my appreciation to my friend who contributed in one way or the other to the
success of this work.
My sincere gratitude also goes to my project supervisor MR NWAKA S. U
whose tolerance and assistance added to the success of this study. My gratitude
also extends to all my lecturers, whose advice and encouragement contributed to
my success in this work.
I am immensely grateful to my Head of Department (HOD) Dr. Nwachukwu
V. N for his encouragement.
 ABSTRACT
This work on the microbial Assessment of freshly caught fish in OtamiriRiver was
assessed in this study using standard microbiological techniques. The results
gotten revealed that the total viable count of the catch fish ranged from
4.4x104cfu/g to 4.10x105cfu/g, with skin being the least while the intestine
recorded the highest. The fin and gill did not recorded any coliform count. The
fungal count ranged from 1.0x10 3cfu/g to 5.2x105cfu/g. The intestine recorded the
highest counts while thefin recorded the least. The total viable count ranged from
1.0104cfu/g to 3.2x105cfu/g. the total coliform count ranged from 0cfu/g to
3.2x104cfu/g. the microorganisms isolated were Staphylococcus aureus,
Micrococcus spp, Streptococcus spp, Bacithis spp, Corynebacterium spp, Proteus
spp, Pseudomonas aeruginosa, Salmonella spp and Escherichia coli.
 TABLE OF CONTENT
Title page i
Approval page ii
Dedication iii
Acknowledgment iv
Abstract v
Table of contents vi
CHARPTER ONE
1.0 Introduction 1
1.1 Aim of the study 4
1.2 Objective of the study 4
1.3 Significance of the study 4
1.4 Scope of the study 4
CHARPTER TWO
2.0 Literature review 5
2.1 Microbial quality of fish and fish product 5
2.2 Fish spoilage
2.3 Microbiological spoilage
2.4 Sources of Microbial contamination in fish water
2.4.1 Food processor/ handling
2.5 Fish related food borne illness and diseases
2.5.1 Staphylococcus Aureus
2.5.2 Pseudomonas Species
2.5.3 Bacillus Species
2.5.4 Escherichia coli
CHAPTER THREE
3.0 Materials and method
3.1 Materials used
3.2 Collection of samples
3.3 Microbiological Analysis of the fish Sample
3.4 Identification of Bacterial Isolates
3.4.1 Gram Staining Techniques
3.4.2 Motility Test
3.4.3 Catalase Test
3.4.4 Citrate Utilization Test
3.4.5 Coagulate Test
3.4.6 Indole Test
3.4.7 Oxidase Test
3.4.8 Sugar Fermentation Test
3.4.9 Spore Staining
3.4.10 Lactophenol Cotton Blue staining Techniques
CHAPTER FOUR
4.0 Results
CHAPTER FIVE
5.0 Discussion Conclusion and Recommendation
5.1 Discussion
5.2 Conclusion
5.3 Recommendation
References

CHAPTER ONE
1.0 INTRODUCTION
Fish is a vertebrate animal, living in fresh and sea water. It is one of the main
sources of animal protein foods available for human consumption (Abdulahi,
2000). Most of the catch comes from oceans, seas, rivers and lately from man-
made ponds. It is highly nutritious food of about 60-80% water 15-25% protein,
11-22% fats, 20% mineral and 1% carbohydrate. It is often cheaper than meat and
also it is a rich protein source for both the poor and the wealthy. Microbial flora
of fish depends on the microbial contents of the water in which they live as the
slime that covers the surface of fish has been found to contain great variety of
bacteria genera. Many dangers, therefore, exist if fish harvested from polluted
water is eaten raw, therefore, exist if the fish harvested from polluted water is
eaten raw, and because of thehigh microbial load of freshly harvested fish it is
susceptible to rapid spoilage. Hence, preservation of fresh fish becomes very
important. This can be achieved by freezing, drying through smoking and sun
drying, canning, etc (Okonkwo, 2001).
Also fish is a very important source of mineral in the diets of man, they constitute
about 60% of the total protein intake in adults especially in the rural areas
(Abolagba, 2006). In Nigeria, fish is eaten cooked, preserved or processed
(smoked) and form a much-cherished delicacy that cuts across socio economic,
age, religious and educational barriers (Adebayo, 2008). Preserving food and
other perishable products like fish and meat generally involves processes that
impede growth inhabiting ingredients or adjusting storage conditions by freezing
or drying. Fish is the most heavily traded food commodity in the market, with the
continuous declining of natural fish production. It is crucial to improve fish
production from aquaculture to world fish production (Gupte and Acosta, 2001).
The production of marketable fish begins with the stocking of stocking of fry into
a rearing environment. These fish can come from wild capture. However, the fish
cannot be guaranteed that adequate numbers can be captured and stocked in the
time corresponding to optimum production condition, the fish farmer then
naturally turn to other means of obtaining his stock which is invariable in artificial
method. (Oyelese, 2006).
Contamination of fish and other fishery products by microbes has been a serious
threat to human health. There are four main factors responsible for fish spoilage
once it is out of its natural habitat (water) and these includes: Autolysis which
usually precedes bacterial spoilage and involves the breakdown of protein and
lipids to amino acids and fats by muscle enzymes. The activity of microorganism is
another factor which uses the amino acid produced by autolysis for proliferation
(Dike et al, 2007) tinned fish products are considered meal compared with other
food meals, easily to be prepared, thus suiting most working ladies and families as
well as canteens and quick service cafeterias. They are also suitable for camping
and other activities where refrigeration may not be available.
Many marine species produce excellent canned products supporting an important
role in the field of human nutrition. During the last decades, there has been
growing interest in determining heavy metal levels in the marine and fresh water
environment, and attention has been drawn to the measurement of
contamination level in public food supplies, particularly fish. Toxicological and
environmental elements in food. The ingestion of food is anobvious means of
exposure to metals, not only because many metals are natural components of
foodstuffs, but also because of environmental contamination and contamination
during processing (Yusuf and El-Shashawi 1999; Ashraf 2006).
Microbial action has been known to play an important role in the spoilage of fish
(Eyo, 2001). Bacterial spoilage characterized by softening of the muscle tissue and
the production of slime and offensive odor (Ames and Paul, 1991). Microbial and
biochemical quality assessment are necessary to ensure the food safety of any
processed product (Azam et al., 2003). Chemical breakdown of protein content,
fat content (agent of rancidity and off-flavor) and the water content or activity is
responsible for spoilage of most fresh and of several lightly preserved seafood’s
(Lund et al., 2000). As earlier reported the microbial populations of fish can vary
significantly (Adebayo-Tayo et al., 2008). A similar report on fish confirmed that,
fish because of their soft tissues and aquatic environment are extremely
susceptible to microbial contamination. Millions of bacteria, many of them
potential spoiler are present on the surface slime, on the gills and in the intestine
of live fish, although the flesh itself is normally sterile. Bacteria growth and
invasion on the fish are prevented by bodies natural defense system during life
but after death the defense system breaks down and the bacterial multiply and
invade the flesh (Abolagba and Uwagbai, 2011)
1.2 Aim of the study
The aim of this project is to assess the microbiological profile associated with fish
(Clarias gariepinus) and Tilapia species caught from Otamiri River, Nekede, Imo
State.

1.3 objectives of study

 To determine the microbial present in freshly caught in otamiri River
 To find the amount of microbial load on the gills, intestine and
skin of fresh water fish
 To determine the possible microbes present in the body of the
fish

1.4 Significance of study

The outcome of this study will help to know the level of microbes on
freshly caught on otamiri River, Nekede Imo State. This was caused as a
result of pollution and human activities in the water. This research will
help for proper handling of fresh product before consumption.

1.5 Scope of Study

This work will include the analysis of microbes on freshly caught fishes
in Otamiri River, using different agar as culture medium and the
incubation of the isolates for 24 hours of incubation, after which
biochemical tests will be run.

CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 MICROBIAL QUALITY OF FISH AND FISH PRODUCT.
Human and microbes have a long history together. The normal flora
consists of organism that makes their home on or in some part of the
body. In a healthy person, such organism rarely cause disease.
Microorganisms of the normal flora may be in symbolic relationship,
where both microorganisms of the normal may be in symbolic
relationship, where both microorganism and host benefit. The enteric
bacteria that form the normal flora of the intestine assist in the
synthesis of vitamin k and some of the vitamins of the B complex. In
commensalism, microorganism are neither beneficial nor harmful to
their host as in the case of the large group of microbial flora that live on
the skin, and the mucous membranes of the upper respiratory tract,
intestines and vagina. Fish is very important foodstuff in developing
countries due to its high content, and nutritional value.
Fish provides more than 50% of the animal protein for the population
of 34 countries (Pal, 2010). However, it spores easily, especially in hot
climates and tropical areas where cold preservation techniques are
often missing. Fish salting or bringing drying or smoking are traditional
techniques for the improvement and storage of fish (Pal, 2010). About
8 million tons of fish (25-30%) of the world catch for human
consumption are dried, salt, smoked or treated by some combination of
these process each year (kamruzzaman, 1992). Fish consist of an
average 70% of water, in fatty fish, and in lean fish about 80%.
When water content of the fish falls below 25% of its net weight,
bacterial action stops, and when it further reduced to 15%, molds cease
to grow. In the dried product with initial moisture content of about
20%, it has to pick up sufficient moisture before any microbial attack.
Fish usually takes 5 to 7 days to dry during, which it gets heavily
contaminated (Doe et al., 1977). Quality of dried fish include
organoleptic and biochemical quality (Azam et al. 2003). Though
seafood is an important element of Mediterranean diets, it plays a
significant role in causing food borne diseases. Fresh seafood is a highly
perishable product; spoilage developing in aerobically stored fish
typically consists of Gram-negative psychrophilic, non fermenting rods.
Thus under aerobic ice storage, the flora is composed almost
exclusively of Pseudomonasspp and Shewanella putrefaciens (chouliara
et al., 2004). The Entire bacteria case count is considered as another
index of fish quality because it is related to storage in ice, washing, and
evisceration (Zambuchini et al., 2008).
Monitoring of these microorganisms has been suggested as a measure
of fish quality. Moreover, risk management decisions should take into
account the whole food chain from primary production of consumption
and should be implemented in the context of appropriate food safety
infrastructures, such as regulatory enforcement, food product tracing
and traceability system. In the fish processing chain, managing risks
should be based on scientific knowledge of the microbiological hazards
and the understanding of the primary production, processing and
manufacturing techniques and handling during food preparation,
storage and transport, retail and catering (Reilly, 2006). Seafood
products contaminated water, which have been improperly preserved
after harvesting are known to play an important role in infections by
Vibrio spp. (Baffone et al,. 2000).
Consumption of raw or undercooked seafood, particularly, shellfish
contaminated with V. Parahaemolyticus may lead to development of
acute gastroenteritis characterized by diarrhea, headache, vomiting,
nausea, abdominal cramps, and low fever (Pal, 2007). This bacterium is
recognized as the leading cause of human gastroenteritis associated
with seafood consumption in the United States, and an important
seafood-borne pathogen throughout the world (Su and Liu, 2007). In
contrast to Asian countries, V. Parahaemolyticus infections are rarely
reported in European countries (Feldhusen, 2000).

2.2 FISH SPOILAGE

Fish spoilage is a complex process, in which physical, chemical and
microbiological mechanisms are implicated (Adebayo-Tayoet al., 2012b,
Pal, 2012). Many spoilage producing bacteria (Aeromonas, Alcaligenes,
Bacillus, Enterobacter, Enterococcus, Escherichia coli, Listeria, Pseudom
onas, Shewanella) and fungi (Asepergillus, Candida, Crytococcus,
Rhodoturula) are isolated from fresh fish and other sea foods (Pal,
2012). Reports on spoilage mechanism and quality assessment of the
storage quality of frozen/chilled tilapia are still not comprehensive (Sil
et al., 2008; Liu et al., 2010; Adebayo-Tayo et al., 2012b). Degradation
of lipids in fatty fish produces rancid odors.
In addition, marine fish and some freshwater fish contain
trimethylamine oxide that is degraded by several spoilage bacteria to
trimethylamine (TMA), the compound responsible for fishy off-odors.
Iron is a limiting nutrient in fish and this favors growth of bacteria such
as pseudomonas that produce siderophores that bind iron. Spoilage is
the result of a series of changes brought about in the dead fishy mainly
due to enzymatic and bacterial action (Pal, 2012). It starts as soon as a
fish is caught and dies. In areas where temperature is high, fish spoils
within 15-20 hours depending on the species and the method of
capture (Adedeji and Adetunji, 2004).
Fish is extremely Perishable commodity due to its high water content
(Pal and Mahendra, 2015). Spoilage is defined as a change in fish or fish
products that renders it less acceptable, unacceptable or unsafe for
human consumption (Pal, 2012). Fish undergoing spoilage has one or
more of the following sign; dis coloration, slime formulation, changes in
texture, off odors, off-flavors, and gas production (Adedeji and
Adetunji, 2004; Pal, 2010). Properties of spoiled fish compared to fresh
fish are strong odor, dark-red gills with slime on them instead of bright
red ones, soft flesh with brown traces of blood instead of a firm flesh
with red blood, and red, milky pupils without slime instead of clear
ones (Pal, 2010).

2.3.MICROBIOLOGICAL SPOILAGE;
Live fish is normally considered to be sterile, but microorganisms are
found in varying numbers on all the outer surfaces (skin and gills) and in
the alimentary tracts of live and newly caught fishes. A normal range of
102-107 (colony forming units)/cm2 on the skin and between 103and
109cfu/g in the gills and intestines has been observed (Adedeji and
Adetunji, 2004). When the aquatic system is contaminated with
pathogenic organisms, these bacteria become part of the shellfish
microflora (Colakoglu et al., 2006), and when consumed with the fish
the hazardous pathogenic Vibrio causes life threatening food borne
infections and possess a considerable public health issue. Pathogenic
vibrio bacteria are represented as important microbial agents of
sporadic and epidemic human infections in the field of food safety
(Espineira et al., 2010).
Most of the organisms found in fishes are associated with food
poisoning infections in humans such as typhoid fever and shigellosis,
whereas the presence of Aspergillus spp reveal possible production of
aflatoxins (Pal et al., 2015). High microbial load of wild catch fish and
wild tilapia was observed due to pollution of the environment which
the fish caught (Emikpe et al., 2011).
The presence of highly pathogenic bacteria such as Bacillus sp.,
Salmonella spp., Shigella spp., E Coli, Pseudomonas spp. And S. aureus is
of public health concern, and indicate possible contamination resulting
from the use of well water. (Pal, 2010)

2.4.SOURES OF MICROBIAL CONTAMINATION IN FISH WATER

Seafood products, especially crustaceans, harvested from
contaminated water or which have been improperly preserved after
harvesting are known to play an important role in infections by Vibrio
spp. (Wafaa et al., 2011). The potential of water to harbor microbial
pathogens and causing subsequent illness is well documented for both
developed and developing countries (Pal, 2013). Water-related diseases
continue to be one of the major health problems globally (Adebayo-
Tayo et al., 2011a; and Anberber, 2014). Okonko et al.,(2008b) reported
that both bacteria and fungi are common flora of frozen fish and fish
related products during packaging. It is estimated that 80% of all illness
are linked to the use of water of poor microbiological quality. Regular
physico-chemical and bacteriological analysis of water at source must
be carried out to determine or check the effectiveness of the treatment
process (Okonko et al., 2008a).
The presence of Staphylococcus auerus, a pathogenic bacterium of
public health concern and important in frozen seafood products, may
possibly contaminate processed frozen seafood products, from source,
as a result of poor handling. Improper hygiene and eventual affect the
health of consumers (Okonko et al., 2008b)

2.4.1 FOOD PROCESSORS/ HANDLERS

Persons serving foods in processing industries may be sources of
microbial inoculation, food poisoning, food intoxication and food
spoilage. A number of organisms including Staphylococcus aureus have
been isolated from the hands of employees working in food
establishments (Pal, 2012; Pal and Mahendra, 2015). Hence, it is
important to mention that any person with purulent skin lesion or
having respiratory infections should not be allowed to work in food
industry (Pal and Mahendra, 2015).

2.5 FISH RELATED FOOD BORNE DISEASES ILLNESS

The subsurface of live, healthy fish is considered sterile, and should not
present any bacteria or other microorganism. On the contrary, as with
other vertebrates, microorganism colonize the skin, gills, and the
gastrointestinal tracts of fish. The number and diversity of microbes
associated with fish depends on the geographic allocation, the season
and the method on the geographic allocation, the season and the
method of harvest.
In general, the natural fish microflora tends to reflect the microbial
communities of the surrounding, waters (Rhea, 2009). The
autochthonous bacterial flora of fish is dominated by Gram-negative
genera including: Acinetobacter, Flavobacterium, Moraxella,
Shewanella and Pseudomonas. Members of the families Vibrionaceae
(Aeromonas spp) are also common aquatic bacterial, and the typical of
the fish flora. Gram-positive organisms such as Bacillus, Micrococcus,
Clostridium, Lactobscillus and Coryneformscan also be found in varying
proportions (Huss, 1995).
Human pathogenic bacterial can be part of the initial microflora of fish,
posing a concern for sea food borne illness (Davis, et al., 2001). These
pathogens can be divided into two groups: organisms naturally present
on fish such as Clostridium Botulinum, pathogenic Vibrio spp.,
Aeromonas spp., and Plesiomonas shigelloides; and those not
autochthonous to the aquatic environment, are present there, as result
of contamination or are introduced to fish during service, processing or
storage (Listeria monocytogenes, Staphylococcus aureus, Salmonella
spp., Shigella spp., Escherichia coli, and Yersinia enterocolitica).

2.5.1 STAPHYLOCOCCUS AUREUS

Staphylococcus aureus is a Gram-positive, round-shaped bacterium that
is a member of the firmicutes, and it is a usual member of the
microbiota of the body, frequently found in the upper respiratory tract
and on the skin. It is often positive for catalase and nitrate reduction
and is facultative anaerobe that can grow without the need for oxygen.
Although S.aureus usually act as a commensal of the human microbiota
it can also become an opportunistic pathogen, being a common cause
of skin infections including abscesses, respiratory infections such as
potent protein toxins, and the expression of a cell- surface protein
resistant strains of S. aureus such as methicillin resistant S. aureus
(MRSA) is worldwide problem in clinical medicine. Despite much
research and development, no vaccine for S. aureus has been approved
(Val et al., 2009).
An estimated 20% to 30% of the human population are long term
carriers S. aureus which can be found as part of the normal skin flora, in
the nostrils, and as a normal inhabitants of the lower reproductive
tracts of women. S aureus can cause a range of illnesses, from minor
skin infections, such as a pimple, impetigo, boils cellutis, folliculitis,
carbuncles, scalded skin syndrome, and abscesses, to life- threatening
diseases such as pneumonia, meningitis, osteomyelitis, endocarditis
toxic shock syndrome, bacteremia and sepsis. It is still one of the five
most common causes of hospital-acquired infections and is often the
cause of wound infections following surgery. Each year, around 500,000
patients in hospitals of the United States contracts a staphylococcal
infection, chiefly by S. aureus.

2.5.2 PSEUDOMONAS AERUGINOSO

Pseudomonas is a genus of Gram-positive, Gamma-protect bacteria,
belonging to the family pseudomonadaceae accounts for 191 validity
described species. The members of the genus demonstrate a great deal
of metabolic diversity and consequently are able to colonize a wide
range of niches. Their ease of culture in-vitro and availability of an
increasing numbers of pseudomonas strain genome sequences has
made the genus an excellent focus for scientific research; the best
studied species include P. aeruginosa in its role as an opportunistic
human pathogen, the plant pathogen P. syringae, the soli bacterium P.
putida, and the plant the plant growth-promoting P. fluorescens.
However, because of their widespread occurrence in water and plant
seeds such as dicots, the pseudomonas were observed early in the
history of microbiology. Bacillus (Latin “stick”) is a genus of Gram-
positive, rod-shaped bacteria, a member of the phylum firmicutes, with
266 named species. The terms is also used to describe the shape (rod)
of certain bactaria, and the plural Bacilli is the name of the class of
bacteria to which this genus belongs. Bacillus species can be either
obligate aerobes: oxygen dependent; or facultative anaerobic: having
the ability to be anaerobic in the absence of oxygen. Cultured Bacillus
species test positive for the enzymes catalase if oxygen has been used
or present (Palleroni, 2010).

2.5.3.BACILLUS SPECIES
Bacillus can reduce themselves to oval endospores and can remain in
the dormant state for years. The endospore of one species from
morocco is reported to have survived being heated to 420 0 C.
Endospore formation is usually triggered by a lack of nutrients: the
bacterium divides within its cell wall, and one side then engulfs the
other. They are not true spores (i.e., not an offspring). Endospores
formation originally defined the genus, but not all such species are
closely related, and many species have been moved to other genera of
the firmicutes. Only one endospore is formed per cell. The spores are
resistant to heat, cold, radiation, desiccation, and disinfectants.
Many species of Bacillus can produce copious amount of enzymes
which are used in various industries, such as the production of alpha
amylase used in starch hydrolysis, and the protease subtilis in used in
detergents. B.subtilis is a valuable model for bacterial research. Some
Bacillus species can synthesize and secrete lipopeptides, In particular
surfactins and mycosubtlilins (Alcaraz et al., 2010)

2.5.4 ESCHERICHIA COLI

Escherichia coli also known as E. coli is a gram-negative facultative
anaerobic, rod-shaped, coliform bacterium of the genus Escherichia
that commonly found. In the lower intestine of warm-blooded
organism (endotherms). Most E. coli strains are harmless, but some
serotypes can cause serious food poisoning in their hosts, and are
occasionally responsible for product recalls due to microbiota of the
gut, and can benefit their host by producing vitamin K2, and preventing
colonization of the intestine with pathogenic bacteria, having s
symbiotic relationship. E. coli is expelled into the environment within
fecal matter.
The aerobic conditions for 3 years massively, but its numbers decline
slowly afterwards. E. coli and other facultative anaerobes constitute
about 0.1% of guts microbiota, and fecal- oral transmission is the major
route through which pathogenic strains of the bacterium cause disease.
Cells are able to survive outside the body for limited amount of time,
which makes them potential indicator organisms to test environmental
samples for fecal contamination. A growing body of research, though,
has examined environmental persistent E. coli which can survive for
and grow outside a host (Alcarazet al., 2010).

CHAPTER THREE
MATERIALS AND METHOD
3.1 MATERIALS USED
Petri-dish, Wire loop, Cotton wool and foil, Round bottom flask, Test-
tube and test tube rack, Spatula, Measuring cylinder, Swap stick, Slide,
Beaker.
EQUIPMENT
Weighing balance, Autoclave, Incubator, Microscope, Hot Air oven.
REAGENTS
SDA, Macconkey agar, Simon citrate agar, Triple sugar Iron agar,
Peptone water, ½ strength Nutrient agar, Cystal violet, lugol’s Iodine,
Safranine, Malachite Green, Hydrogen peroxide.

3.2 COLLECTION OF SAMPLE

The fish sample (Clariasgariepinus and Tilapia species) were collected
from OtamiriRiver in Owerri west and taken to the Researchers
laboratory in Umuerim extension for microbiological analysis.

3.3 MICROBIOLOGICAL ANALYSIS OF THE FISH SAMPLES A swab stick

was used to scrub each of the fish samples and separately placed in
nine milliliter (9ml) of sterile water contained in a test tube and serially
diluted using ten-fold serial dilution for each treatment sample.After
the serial dilution, 0.1ml of the 10-3 dilution was aseptically inoculated
onto sterile plates of Nutrient agar, MacConkey agar and Sabroid
Dextrose agar standard culture media. They were incubated at 37 oC for
24 hours for the bacteriological media and at 27 oC for 48hours for the
mycological media. After the incubation periods, the microorganisms
that grew on the culture plates were counted using the colony counter.

The microbial isolates obtained were thereafter identified using cultural

morphology. The bacterial isolates were further characterized using
gram staining and biochemical tests while the fungal isolates were
further characterized using lacto phenol cotton blue staining
techniques.
3.4. IDENTIFICATION OF BACTERIAL ISOLATES

The bacterial isolates from the plates were identified by gram staining
and biochemical tests.

3.4.1 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 therefore. The smear 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 40 oil immersion objective lens (Cheesbrough,
2005). Gram positive bacteria gave purple coloration while gram
negative bacteria gave pinkish coloration.

 3.4.2 MOTILITY TEST

SulphideIndole Motility (SIM) agar was used in this test. Each of the test
organisms was aseptically inoculated into sterile tubes of SIM agar
using stab inoculation method. The level where the inoculation stopped
was noted in each case of the test organisms and therefore incubated.
Atthe end of the incubation period, an extension in the level of the
inoculated tubes was noted in each case of the test organism. The
tubes that had extension in growth through the line of inoculation were
recorded as positive while those that had none were recorded as
negative (Anderson, 2000).

3.4.3 CATALASE TEST

This test is used to differentiate those bacteria that produce the

enzyme catalase such as staphylococci from non-catalase producing
bacteria such as streptococci. About 2ml of hydrogen peroxide solution
was poured into several test tube for each of the bacterial isolates.
Using a sterile wooden stick, each colony of the bacterial isolates was
immersed in each of the hydrogen peroxide solution. Active bubbling
within 10 seconds is an indication of a positive test while none is an
indication of a negative test (Cheesbrough,2005).

3.4.4 CITRATE UTILIZATION TEST

This test helps in the identification of entrobacteriaceae.Each of the

test organisms were inoculated into sterile agar slopes of simmon
citrate agar in each case using stab inoculation techniques. The
inoculated agar slopes were then incubated at 37 0C for 24hours. A
bright blue coloration is an indication of a positive test while none is an
indication of a negative test (Cheesbrough, 2005).

3.4.5 COAGULASE TEST

A drop of distilled water was place on each of the organisms. Thereafter

a colony of each of the test organisms was emulsified in e4ach of the
drops to make a thick suspension. A loopful of plasma was then added
to one of the suspension and mixed gently for each of the test
organisms. Clumping of the organism within 10 seconds was an
indication of a positive test while none was an indication of negative
test (Cheesbrough, 2005).

3.4.6 INDOLE TEST

Some microorganisms are capable of hydrolyzing the amino acid

Tryptophan and one of the end products is indole. The ability of a
microbe to carry out this reaction can be used for biochemical
characterization. The test organisms were suspended in sterile peptone
(about 3ml) preparation in sterile test tubes incubated at 37 0C for
48hours after which 0.5ml of Kovac’s reagent was added and shaken
gently. A red coloration in the surface layer within 10 minutes is an
indication of a positive test while none is an indication of a negative
test (Cheesbrough, 2005).

3.4.7 OXIDASE TEST

The method of Cheesbrough (2005) was adopted for this test. A piece
of filter paper was placed in a clean petri-dish and three drops of fleshly
prepared oxidase reagent was added in each case of the test organisms.
With a sterile piece of stick, each colony of the test organisms was
removed and smeared on each oxidase reagent drop on the filter
paper. The development of a blue-purple coloration is an indication of a
positive test while none is an indication of a negative test.

3.4.8 SUGAR FERMENTATION TEST

Each colony of the different test organisms were inoculated onto sterile
agar slopes of triple sugar iron agar using stab inoculation. After this,
the inoculated, agar slopes were incubated at 37 0C for 24hours. The
different color’s of the scopes and butts in addition to the presence of
gas production and hydrogen sulphide (H 2 S) blackening is indicative of
the type of bacteria present (Cheesbrough, 2005).

3.4.9 SPORE STAINING

A smear of each of the bacterial isolates was made on a clean grease-

free slide and fixed by air drying. The smears were then covered with
malachite green and place over steam for 5 minutes while topping the
slides with more malachite green when they are dried out. At the end
of 5 minutes, the smears were washed off with clean water and counter
stained with Safranin for 2 minutes and wash off with water. The
smears were allowed to dry before they were viewed with x 40 oil
immersion objective lens (Fawole and Oso, 2004). Spore positive slides
gave a co-appearance of pink and green color while negative slides gave
only pinkish coloration.

3.4.10 LACTOPHENOL COTTON BLUE STAINING TECHNIQUE

The fungal isolate were identified by morphological characteristics on

Saboraund Dextrose Agar (SDA) and microscope examination after
lactophonol cotton blue staining technique.

Each of the fungal isolates were separately collected with a sterile

wooden stick and teased out on a drop of lactophenol cotton blue stain
on a clean glass slide. The wet mount preparation were then viewed
under the microscope for branched and unbranched hyphae (Fawole
and Oso, 2004).
 CHAPTER FOUR

4.0 RESULTS

The results of the microbial load of the fish samples caught from the
Otamiri River in NekedeOwerri, Imo state are presented in Table 1 and
Table 4.3(Clarias and Tilapia respectively) .,Table 2 is the result of the
Morphological and Biochemical Characteristics of Bacterial isolates
from the Fish samples.

Table 4.1: Total Microbial load of the fish samples (Clariasgariepinus)

caught from the OtamiriRiver, NekedeOwerri.

Samples total viable total coliform Total fungal

count (cfu/g) count (cfu/g) count (cfu/g)

 Intestine 4.10x105 8.0x103 5.0x103

Fin 1.00x105 ------ 1.0x103

Skin 4.4x104 2.0x103 3.0x103

Gills 1.04x105 ------ 4.2x103

Table 4.3: Total microbial load of the fish samples (Tilapia) caught from
the Otamiri River, Nekede, Owerri.

Samples total viable total coliform Total fungal

count (cfu/g) count (cfu/g) count (cfu/g)

Intestine 3.20x105 6.0x103 7.2x103

Fin 1.14x105 3.2x104 1.0x103

Skin 2.1x104 ______ 2.0x103

Gills 1.0x105 _____ 1.0x103

 CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 DISCUSSION

The result of the microbial load of fish samples caught from otamiri as
presented in the table 4.1 revealed that the total viable count (TVC) of
the catfish parts ranged from 4.4x104 cfu/g to 4.10x105 cfu/g., the skin
being the least while the intestine recorded the highest., the coliform
count ranged from 0 cfu/g to 8.0x10 3 cfu/g., the fungal count ranged
from 1.0x103 cfu/g to 5.0x103 cfu/g., the intestine recorded the least .
The total viable count of the Tilapia fish as recorded in table 4.1
revealed that the total viable count ranged from 1.0x10 4 cfu/g to
3.20x105 ., the total coliform count ranged from 0 cfu/g to 3.2x10 4
cfu/g., the fungal counts ranged from 1.0x10 3 cfu/g to 7.2x105 cfu/g.
The intestine also recorded the highest fungal counts. The catfish
generally had higher microbial load than the Tilapia. This can be
attributed to the larger surface area of catfish compared to Tilapia fish.

The bacterial isolates obtained in this work were Staphylococcus

aureus, Micrococcus spp, Streptococcus spp, Bacillus spp,
Corynebacteriun spp, Proteus spp, Pseudomonas aeruginosa,
Salmonella spp, and Escherichia coli. Budiati et al., (2015) also reported
the microbial quality of clariasgariepinus and Tilapia. In their study, the
reported microbial load of catfish to be in the tone of 10 6cfu/g as the
total viable count. , 102 to 103 as the total coliform with count of the
tilapia and 101 cfu/g as the total fungal count while tilapia recorded
lowest microbial load when compared with the catfish. This is in line
with the outcome of this work.

The presence of E. coli indicates recent feacal contamination of the

water body. The presence of theseorganism is of really of public health
concern.

5.2 CONCLUSION
This work has shown that Tilapia and Catfish habors microorganisms of
public health importance. Tilapia had lower microbial load compared to
the catfish samples perhaps due to the lower surface area of Tilapia.
The presence of these microorganisms indicates poor water quality and
is of public health concern since they can cause disease in man.
5.3 RECOMMENDATIONS
The outcome of this work has necessitated the following
recommendation:
 That fresh fish samples should be properly cooked to reduce
their microbial load and reduce cases of food-borne disease.
 That people should stop dumping untreated wastes including
sewage to reduce its microbial load.
 That the government should regulate the dumping of refuse in
rivers to prevent eutrophication.

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