ISOLATION, CHARACTERIZATION AND IDENTIFICATION OF MICROORGANISMS PRESENT IN SMOKED TILAPIA AND MACKEREL FISH

ABSTRACT

This study focuses on the Isolation, Characterization, and Identification of Microorganisms Present in Smoked Tilapia and Mackerel Fish sold in Umudike, Abia State, Nigeria. The primary objectives are to isolate and identify microorganisms present in the fish samples and assess whether they are safe for consumption. Smoked fish samples were collected from two different markets in Umudike: Gate 6 and Amaoba. Samples were collected aseptically and transported to the Microbiology Laboratory at Michael Okpara University of Agriculture for microbiological analysis. Standard techniques, including sterilization of materials and media preparation, were employed to ensure aseptic conditions. Nutrient agar and Sabouraud Dextrose agar (SDA) were used for culturing bacteria and fungi, respectively.

Microbiological analysis involved the preparation of fish samples, serial dilution, and inoculation of plates. Colony enumeration and isolation of bacteria and fungi were performed after incubation. The isolated organisms were characterized through various tests, including Gram staining, motility, catalase, oxidase, and sugar fermentation. Confirmatory identification was conducted using Bergey's Manual of Determinative Bacteriology.

The results revealed the presence of several bacteria, including Staphylococcus aureus, Bacillus species, Pseudomonas aeruginosa, Streptococcus species, and Escherichia coli. The highest bacterial load was found in tilapia fish, with Staphylococcus aureus being the most prevalent organism, accounting for 41% of the isolates. Fungal isolates included Aspergillus and Fusarium species, with Aspergillus being the dominant fungus in both fish types.

The study concludes that smoked tilapia and mackerel fish sold in Umudike are contaminated with spoilage organisms, as well as potentially pathogenic bacteria and fungi. The presence of Staphylococcus aureus, a known foodborne pathogen, raises public health concerns, emphasizing the need for improved hygiene during fish processing and handling. To ensure food safety, stricter environmental regulations and education on personal hygiene for food handlers are recommended. Additionally, consumers should properly cook smoked fish before consumption to prevent foodborne illnesses

TABLE OF CONTENTS

CHAPTER ONE

1.0     Introduction

1.1       Aims and Objectives

CHAPTER TWO

2.1     Literature Review

2.2.1  Tilapia Fish

2.2.2  Characteristics of Tilapia Fish

2.2.3  Species of Tilapia

2.2.4  Aquarium Species

2.2.5  Tilapia as Food

2.2.6  Mackerel Fish

2.2.7  Sombroid Mackerels

2.2.8  Scombrini, the True Mackerels

2.2.9  Other Mackerel

2.2.10  Characteristics of Mackerel

2.2.11  Life Cycle Of Mackerel

2.2.12  Mackerel as Food

2.3     Bacteria Transmissible To Human Being through Contact with Fish

2.3.1  Mycobacterium Spp.

2.3.2 Streptococcus Iniae;’

2.3.3  Erysipelothrix Rhusiopathiae

2.4     Foodborne Pathogens Associated With Fish to Humans

2.4.1  Vibrio Parahaemolyticus and Other Vibrios

2.4.2  Vibrio Cholerae

2.4.3  Escherichia Coli

2.4.4  Aeromonas Spp.

2.4.5  Salmonella Spp.

2.4. 6 Staphylococcus Aureus

2.4.7  Listeria Monocytogenes

2.4.8  Clostridium Botulinum

2.4.9  Clostridium Perfringens

2.4.10 Campylobacter Jejuni

2.4.11  Shigella Spp.

2.5     Histamine Fish Poisoning

2.6     Overview of Sources and Routes of Contamination of Fish Consumed By Humans

2.6.1  Raw Material

2.6.2  Processing Equipment

2.6.3  Personnel

2.6.4 Water

CHAPTER THREE

3.0     Materials and Methods

3.1     Study Area

3.2     Collection of Samples

3.3     Sterilization of Materials

3.4     Media Used

3.5     Microbiological Analysis

3.5.1 Sample Preparation

3.5.2 Preparation of Serial Dilution

3.5.3 Inoculation of Plates

3.6     Characterization of Bacterial Isolates  

3.6.1  Identification Fungal Isolates

3.6.2  Gram Staining              

3.6.3  Procedure

3.6.4  Catalase

3.6.5  Coagulase Test

3.6.6  Citrate Test

3.6.7 Indole Test

3.6.8  Sugar Test

CHAPTER FOUR

RESULTS

CHAPTER FIVE

DISCUSSION

Conclusion

References

APPENDIX I: ANOVA Table for Bacterial load

APPENDIX II: ANOVA Table for Fungal load

LIST OF TABLES

Table 1:           Biochemical and microscopic characteristics of bacteria isolated

Table 2            Colony morphology, Microscopic feature and Fungal Isolate

LIST OF FIGURES

Figure 1: Bacteria Count of Smoked Tilapia and Mackerel Fish Sold in Gate 6 market

Figure 2: Bacteria Count of Smoked Tilapia and Mackerel Fish Sold in Amaoba market

Figure 3: Fungal Count of Smoked Tilapia and Mackerel Fish Sold in Gate 6 market

Figure 4: Fungal Count of Smoked Tilapia and Mackerel Fish Sold in Amaoba maket

Figure 5: Percentage Occurrence of Smoked Tilapia and Mackerel Fish Sold in both         

               markets ( Gate 6 and Amaoba markets )

Figure 6: Percentage Occurrence of Smoked Tilapia and Mackerel Fish Sold in both        

              markets (Gate 6 and Amaoba markets )

CHAPTER ONE

1.0       INTRODUCTION

Fish is a vertebrate animal, living in fresh and seawater. 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 (Adams and Moss, 1999). It is a highly nutritious food of about 60-80% water, 15-25% protein, 11-22% fat, 20% mineral and 1% carbohydrate (Uzuegbu and Eke, 2000). It is often cheaper than meat and so it is a rich protein source for both the poor and the wealthy. Microbial flora of fish depends on the microbial content 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 (Okonkwo, 2001). Many dangers therefore exist if fish harvested from polluted water is eaten raw, and because of the high 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. Smoking simply means a heating process that dries the fish to preserve it from spoilage (Olokor et al., 2007). Most dried fish consumed in Nigeria are smoked (Dike et al., 2007). Smoking of fish from smoldering wood for its preservation dates back to civilization (Olokor et al., 2007). The steps in the smoking process are necessary not only for safe preservation, but also to produce good flavor and aroma (Ray and Ray, 2004). Hence smoked fishes are less prone to microbial spoilage than fresh fish. However spoilage still occurs as a result of growth of microbes due to partial dehydration during smoking.

Fish is widely consumed as a remarkable source of protein in Africa. In riverine communities such as Niger delta communities of Nigeria, fish is more widely consumed than meat (Obiajuru and Ogbulie, 2006). Recent studies in Owerri, Imo State showed 20% prevalence of salmonellosis in frozen fish (Ohalete et al., 2011). The origin of most microbes in fish and fish products may not be precisely known, some workers have related microbial infections and/or contamination of fish and fish products to a number of factors including: unfavorable conditions in the fish culture system (Horsley, 1973;Okpokwasili and Ogbulie, 1993), pollution and seasonal changes (Obiajuru, et al., 2010), fish handling and processing including personnel and processing equipment (Pelczer and Chan., 1986). Fish contains protein and nutrients favorable for microbial attack even after processing. This often leads to fish spoilage and nutritional deterioration.

The advantages of fish as a food are its easy digestibility and high nutritional value (Leisner et al., 2001). These important attributes makes the commodity readily susceptible to microbial attack particularly bacteria (Adams et al., 1999). Fish flesh naturally contains very low levels of carbohydrates and these are further depleted during the death struggle of the fish (Adams et al., 1999). This has two important consequences for spoilage. Firstly, it limits degree of post mortem acidification of the tissue so that the ultimate pH of the muscles is 6.2-6.5 (Adams et al., 1999).Disease breaks out in fish tank very quickly and you have to first identity the type of disease before you can take action.

The bacteria are transmitted by fish that have made contact with other diseased fish. Bacterial fish disease and infections are very common and are one of the most difficult health problems to Deal with (Douglas, 2007). Bacteria can enter the fish body through the gills or skin or it can stay on the surface of the body (Douglas, 2007). There are four types of bacterial infections. Bacterial gill disease: The gills are the primary target, Systemic bacterial disease: bacteria invades the fish’s body and damages internal organs, bacterial body ulcers: Lesions on the fish body that can be shallow or deep and fin rot: Most likely resulting from environmental stress (Douglass, 2007). Secondly, the absence of carbohydrate means that bacteria present on the fish will immediately resort to using the soluble pool of readily assimilated nitrogenous material, producing off-odour (Adams et al.,1999). 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 include: 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 (Onamiwo, 1998). Others are chemical deterioration and insect attack which cause considerable deterioration. However, spoilage of fresh and highly preserved fish products is mostly caused by microbial action. Foods of high sugar/salt contents are therefore most likely to be spoiled by any kind of microbe (Abey, 2007). It has been reported that serious disease outbreak had occurred in both man and animals after consuming some dried fish feed and food (Dike et al., 2007). This could be as a result of disease causing microorganism like Escherichia coli, Salmonella typhi, Vibrio cholerae which results from poor handling/cross-contamination and improper processing practice of ready to eat “smoked fish” products. Other microorganisms of primary concern are Listeria monocytogenes and Clostridium botulinium. Extensive handling provides opportunities for other food borne pathogens to contaminate products if sufficient attention is not given during smoking process (Eklund et al., 1993). Tilapia and mackerel are one of the most sold and consumed fish in umudike.

1.2           AIMS AND OBJECTIVES

                        i.         To isolate, characterize and Identify the microorganisms present in smoked tilapia and mackerel fish sold in Umudike.

                       ii.         To analyze and identify whether the two smoked fishes sold in the market is good enough for consumption.

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