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					   MICROBIAL PROFILE OF SUYA MEAT IN ENUGU STATE




                             BY



                 IBE ROSEMARY NKECHINYERE

                         MB/2006/154



      DEPARTMENT OF MICROBIOLOGY AND BIOTECHNOLOGY

                     CARITAS UNIVERSITY

                     AMORJI NIKE ENUGU




                        AUGUST 2010.




MICROBIAL PROFILE OF SUYA MEAT IN ENUGU STATE


                             BY
                                                            2

                IBE ROSEMARY NKECHINYERE
                        MB/2006/154


               A PROJECT SUBMITTED TO THE
DEPARTMENT OF MICROBIOLOGY AND BIOTECHNOLOGY, FACULTY OF
                   NATURAL SCIENCES,
                   CARITAS UNIVERSITY,
             AMORJI –NIKE, ENUGU, ENUGU STATE.




 IN PARTAL FULFILMENT OF THE REQUIREMENT OF THE AWARD OF
    BACHELOR OF SCIENCE (B.SC), DEREE IN MICROBIOLOGY AND
                      BIOTECHNOLOGY.



                       AUGUST, 2010.
                                                                                              i

                                     CERTIFICATION

This is to certify that the project titled “Microbial Profile of Suya Meat in Enugu State,” was

carried out by Ibe Rosemary Nkechinyere under the supervision of Ms Nmema E.E. and it is

accepted in partial fulfillment of the Bachelor of Science (B.Sc) degree in of Microbiology

and Biotechnology. The department recognizes that Ibe Rosemary Nkechinyere

(MB/2006/154) bear full responsibility for the content of this work.


________________                                            ____________
IBE ROSEMARY N.                                                  Date
________________                                            _____________
Miss Nmema E.E.                                                  Date
(Supervisor)
________________                                            _____________
Mr Amadi E.C.                                                     Date
(H.O.D.)
________________                                            _____________
Prof. Nduka Okafor                                                Date
(Dean of Natural science)
___________________                                           ____________
PROF. J.C. OGBONA
External Examiner                                                  Date




                                       DEDICATION
                                                                                     ii

I dedicate this work to God Almighty and to our Mother Mary for the grace and strength

given to me to carry out this work successful




                                ACKNOWLEDGEMENT
                                                                                            iii

My profound gratitude goes to God the father, who has been a father to me; the son who has

been a true friend and to the Holy Spirit who has been a brother and my companion

throughout my years as an undergraduate.


  My special thanks goes to my project supervisor Ms Nmema E.E., whose untiring support,

suggestion, and guidance has led me this far and made this research writing worthwhile.


I am particularly grateful to Prof. Nduka Okafor (Dean of Natural Science), Mr Amadi

E.C.(H.O.D) and to all my lecturers who have done a tremendous work in my academic

pursuit, I pray that God Almighty will bless you all in million fold.


My sincere gratitude also goes to all the members of my family; Mr and Mrs Ibe who

worked so hard for me both morally and financially, I pray that God will give them long

lives and good health to enjoy the fruit of their labour. Also to my siblings especially to my

dearest elder sister Mrs Ibeh Kelechi whose encouragement, financial support, and love have

kept me going throughout my stay in school; and to my lovely brother Ikechukwu and sisters

Chika, Ogechi, Chiamaka and Chichi. I will not forget my relatives, aunty Elizabeth, aunty

Susan, aunty Ann and others, I want to say a big thank you for always being there for me. I

love you all.


My appreciations also go to my darling friends who have always being there to pray, finance

and encourage me when I felt that all hope was lost; they are Juliet, brother Christian,

Everistus Gabriel, Charles Oche, sister Happiness, Chioma, Blessing, brother Kenneth

Titilope,Nonye, Bolanle,to mention but a few.
                                                                                         iv

I will not forget to thank myself for typing a beautiful work and all who assisted and

corrected me in one way or the other in the course of my typing.




                            ABSTRACT

Twenty (20) samples of suya meat in Enugu were collected randomly and analyzed

microbiologically and the isolates were identified as Staphylococcus aureu (35%),

Escherichia coli (15%), Streptococcus species (15%), Pseudomonas (35%). The most

frequently isolated organisms were Staphylococcus and Pseudomonas. The total viable

bacterial counts ranged from 1.9x        3.8x    cfu/g whereas, total coliform count ranged
                                                                                              v

from 1.1x        -3.0x       cfu/g on Nutrient agar and MacConkey agar respectively. The result

revealed that the hygienic condition of the meat was below acceptable standard for human

comsumption.




                                         TABLE OF CONTENTS

Certification -          -     -     -      -    -     -     -     -i


Dedication- -            -     -     -      -    -     -     -     -ii


Acknowledgement-               -     -      -    -     -     -     -iii


Abstract-    -           -     -     -      -    -     -     -     -iv


Table of contents- -           -     -      -    -     -     -     -v
                                                                          vi

List of Tables-     -      -   -      -     -      -     -      -vi


CHAPTER ONE- -             -   -      -     -      -     -      -1

1.0 Introduction- -        -   -      -     -      -     -      -1

1.1 Back ground of study-      -      -     -      -     -      -1


1.2 Aim And Objectives -       -      -     -      -     -      -4


CHAPTER TWO-               -   -      -     -      -     -      -5

2.0 Literature review-     -   -      -     -      -     -      -5


2.1Suya meat-       -      -   -      -     -      -     -      -5


2.2 Preparation of suya- -     -      -     -      -     -      -5


2.3 Microbiology of meat-      -      -     -      -     -      -6    -


2.4 Meat spoilage- -       -   -      -     -      -     -      -7


2.5 Factors that affect the growth of microorganisms in meat-   -8


2.5.1 Temperature--        -   -      -     -      -     -      -8


2.5.2 pH-    -      -      -   -      -     -      -     -      -9


2.5.3. Water availability --   -      -     -      -     -      -9


2.5.4 Nutrients-    -      -   -      -     -      -     -      -10


CHAPTER THREE-             -   -      -     -      -     -      -11
                                                              vii

3.0 Materials and methods-      -     -     -   -   -   -11

3.1 List of reagents-     -     -     -     -   -   -   -11


3.2 List of glassware-    -     -     -     -   -   -   -13


3.3 Preparation of media- -     -     -     -   -   -   -14


3.4 Sample collection -   -     -     -     -   -   -   -14


3.5 Pretreatment of samples-    -     -     -   -   -   -15


3.6 Determination of total viable counts-   -   -   -   -15


3.6.1 Gram reaction-      -     -     -     -   -   -   -16


3.6.2 Motility test- -    -     -     -     -   -   -   -17


3.6.3 Catalase test -     -     -     -     -   -   -   -17

3.6.4 Coagulase test -    -     -     -     -   -   -   -18


3.6.5 Oxidase test- -     -     -     -     -   -   -   -18


3.6.6 Urease test- -      -     -     -     -   -   -   -19


3.6.7 Citrate test - -    -     -     -     -   -   -   -19


3.6.8 Vogues Proskauer test -   -     -     -   -   -   -20


3.6.9 Indole test- -      -     -     -     -   -   -   -21


3.6.10 Carbohydrate fermentation test-      -   -   -   -21
                                                          viii

3.6.11 Methyl red test-   -   -   -   -   -   -     -22

CHAPTER FOUR-             -   -   -   -   -   -     -23


4.0 Results -     - -     -   -   -   -   -   -     -23


CHAPTER FIVE-             -   -   -   -   -   -     -29


5.1 Discussion-     -     -   -   -   -   -   -     -29


5.2 Summary and Conclusion- -     -   -   -   -     -31


5.3 Recommendations - -       -   -   -   -   -     -32


   References-      -     -   -   -   -   -   -     -33


Appendix- -         -     -   -   -   -   -   -37
                                                                     ix




                                  LIST OF TABLES


TABLE 1: Total viable and coliform counts -     -     -   -   -25


TABLE 2: Characterization /identification of Isolate- -   -   -26

TABLE 3: Frequency of occurrence of isolates- -       -   -   - 28
                                                                                                     1

                                            CHAPTER ONE


      1.0                           INTRODUCTION


1.1         BACKGRONUD OF THE STUDY

             Meat is the flesh of animals which serves as food; it is obtained from sheep, cattle,

      goat and swine (Haman, 1997). Meat is a major source of protein and an important source of

      vitamins for most people in many parts of the world, thus they are essential for the growth,

      repair and maintenance of body cells which is necessary for our everyday activities.

             Meat could be traced back to human history, then when primitive men use raw flesh of

      dead animals. But as man developed, he domesticated wild animals. Beef have been the

      major supply of meat in Nigeria as a result of extensive and semi-intensive cattle production

      system in Nigeria by Fulani and Hausa people of the northern Nigeria. (Umoh, 2004).

        Suya meat is a boneless lean meat of mutton, beef, goat or chicken meat staked on sticks,

      coated with sauces, oiled and then roasted over wood using a fire from charcoal. It is a

      traditionally processed meat product and is usually not done with strict hygiene condition

      because they are still done locally.that is served hot and sold along streets, at clubs, picnics

      centers, and restaurants and within institutions. Suya meat is one of the intermediate

      moisture products that are easy to prepare and highly relished.

        Due to the chemical composition and characteristic, meat are highly perishable foods

      which provide an excellent medium for growth of many hazardous microorganisms that can

      cause infection in human and also lead to meat spoilage and economic loss. The most

      important bacterial meat spoilage is caused by lactic acid bacteria which is physiologically
                                                                                            2

related group of fastidious and ubiquitous gram-positive organisms. These include many

species such as Lactobacillus, Leuconostoc, Pediococcus and Streptococcus.

  Since meat has a high nutritive value, microorganisms could easily grow on it. The

possible sources of contamination are through slaughtering of sick animals, washing the

meat with dirty water, handling by butchers, contamination by flies, processing close to

sewage or refuse dumps environment, spices, transportation and use of contaminated

equipment such as knife and other utensils. (Igyor and Uma, 2005).

  The slaughtering process affords extensive contamination of sterile tissue with gram-

negative enteric bacteria from animal intestine including Salmonella species and Escherichia

coli as well as contaminant such as gram-positive Lactic cocci associated with humans,

animals and the environment. Enterococci and Clostridia have been isolated from lymph

node of red meat animals (Lawries, 2000, Alexander et al. 1998).

  Microorganisms grow on meat causing visual, textural and organoleptic changes when

they release metabolite (Jackson et al. 2001). The smoke produced as a number of effects

including preservative effect resulting from the deposition of organic compounds all presents

in the smoked product (Suya meat). (Dineen et al.1999). A preservative effect is also

induced by the surface drying that occurs to the extent of 30% total weight loss in hot

smoked product. Antioxidant effect is produced by the phenolic deposite unto the product.

   The microbial load in meat and meat product increases as long as growth conditions are

favorable. The factor influencing microbial growth includes acidity, pH, temperature, water

activity. Gaseous requirement, nutrient and competition of microbes for the nutrient.

Controlling these factors implies maintaining long shelf life of meat and meat product but
                                                                                               3

proper preservation of meat could be achieved by the combination of two or more

preservation method which includes drying, salting and high temperature (Nester et al.

2001).




 1.2               AIMS AND OBJECTIVES

       This work is aimed at determining the microbial quality of suya meat sold in Enugu

and has the following objectives:

1.       To   isolate,   characterize   and   identify   microbial   species   associated   with

suya meat

2. To establish the public health implication of consumption of suya meat.

3. To offer useful information where necessary to the consuming public.
                                                                                             4




CHAPTER TWO


     2.0   LITERATURE REVIEW

     2.1    SUYA MEAT

      Suya meat is a traditional stick meat product that is commonly produced       by    the

Hausas in West Africa from beef, although chicken can also be used. It is produced from

boneless meat hung on stick and spiced with        peanut cake, salt, vegetable oil and other

flavor, followed by roasting    around a glowing charcoal fire. The smoke from the fire has

a     preservative effect on the Suya meat (Ogunbanwo et al. 2004).


2.2 PREPARATION OF SUYA

      Usually in Nigeria, skinless, boneless flesh of cattle or chicken is used for commercial

preparation of suya meat. Finely grounded roasted peanut cake, red pepper, salt, grounded

ginger, grounded garlic, chunked fresh tomatoes and minced fresh onions are required on
                                                                                              5

suitable conditions depending on the quality of suya meat required to be prepared(Jay,

2000;Judge et al.2000).


      The process of preparation involves a few steps, first is the    grounding of peanut.

The shell and the skin are removed from the peanut before grinding into fine powder using

mortar and pestle or crushed with a rolling pin. If the powder is oily; it is wrapped with an

absorbent paper and squeezed for a minute or two. Next, the grinded pepper, garlic, ginger

are stirred into the peanut powder and mixed properly.

      The meat is then cut into small sizes or thin sliced, dipped and rolled in a bowl

containing the mixed peanut-spice and allowed to coat completely. The minced o meat are

then kept for thirty minutes or more for the peanut cake to stick to it after which the meat

slices are threaded unto skewer and brushed with vegetable oil and roasted on the glowing

charcoal fire for fifteen to twenty minutes. It is finally removed from the skewer and served

hot in a newspaper with sliced onions, tomatoes and cabbage (Judge et al.2000).


2.3     MICROBIOLOGY OF MEAT.

      During slaughtering process, there is contamination of the sterile tissue with intestinal

flora such as gram-negative organisms which includes Escherichia coli as well as

contaminants such as Pseudomonas specie and gram-positive lactic acid bacteria and

Staphylococci species associated with humans, animals and their environment. Meat spoilage

is usually associated with gram-negative proteolitic bacteria which literally decompose the

protein with production of offensive odour (Hamman, 1997).
                                                                                            6

      The addition of salt and drying of fresh meat have been an effective means to control

the meat microflora and thus preserve the tissue for later consumption. The curing salt

(sodium chloride or sodium nitrate or sodium nitrite) and subsequent proper handling

methods, favours the growth of gram-positive bacteria, primarily Staphylococcus aureus

while inhibiting the proliferation of gram-negative bacteria (Boles et. al. 2000).

      There are also other types of common microorganisms apart from enteric organisms

found in meat which are members of Micrococcaceae and Staphylococcaceae families. The

predominant types are coagulase-negative Stapylococci that are salt tolerant and can also

grow with or without oxygen. The most common strain belongs to the species of

Stapylococcus carnosus, S. xylosus and S. kocuria. However, these organisms are harmless

and do not present a microbial hazard. The most common lactic acid microorganisms found

in fermented meat are various strains of Lactobacilli, Leuconostoc, Pediococci, Streptococci

and Enterococci (Lawries, 2001).

      Bacillus species, Staphylococcus aureus, Staphylococcus epidermidis, Proteus species,

Serratia species and Aspergillus species were isolated from suya meat samples collected

from Enugu State (Chukwura and Mojekwu, 2002).


2.4    MEAT SPOILAGE

      Meat is spoilt when it loses its nutritive value, texture and brings out offensive odour

thus rendering it unfit for human consumption. A number of factors could cause meat

spoilage (Nester et al.2001).
                                                                                                7

      Most deterioration or spoilage of meat could be caused by bacteria, yeast and mould.

When meat is not properly handled, it leads to microbial spoilage which makes it unfit for

human consumption (Sokari et al. 1999).


2.5    FACTORS THAT AFFECT THE GROWTH OF                         MICROORGANISMS ON

MEAT.

       A lot of factors affect the growth of microorganisms on meat. These factors include

temperature, pH, water availability, presence of nutrients, moisture, acidity (intrinsic

factors), gaseous requirement, atmosphere of storage(extrinsic factors) (Nester et al. 2001).


2.5.1 TEMPERATURE

      Microorganisms have optimum, minimum and maximum temperature that they can

grow. Listeria monocytogenes have been found to grow at          C and even survive freezing.

This ability of growing at low temperature provides opportunity for proliferation in

contaminated meat products (Fraizer and Westhoff, 2000). Pseudomonas species grow at

temperature less than 20 and so are found.(Dineen et al.1999).


Psychrophiles have optimum temperature between         C and     C, mesophiles between      C

and     C and thermophiles from       C or     C (Fararatti, 2000).


Examples of psychrophiles are


1.    Achromobacter

2.    Alcaligens

3.    Pseudomonas
                                                                                               8

4.    Streptococcus

5.    Salmonella

6.    Most yeast

7.    Mould.


Examples of mesophiles are Bacillus strearothermophiles (Evans and Niren, 1980).


2.5.2 pH

     Most bacteria grow optimally at above pH 7 and not well below pH 4 or above pH 9

(Abdul Raouf et al. 1995). But the pH of maximal growth is determined by the simultaneous

operation of variables other than the degree of acidity or alkalinity. Proteolitic enzymes

operate best near neutral pH 7.


2.5.3 WATER AVAILABILITY

     Water is required by microorganism so reducing water below the optimum level will

prolong shelf life of meat. If meat is stored at a relative humidity below 95%, moisture will

be lost from the surface. Since most spoilage bacteria can grow only on the surface, drying

the meat. Surface will not favor their growth but Staphylococcus aureus can grow in meat

with 0.86%, which is lower than that of other spoilage bacteria (Nester et al. 2001). Moulds

are able to grow in drier conditions than bacteria so that desiccation has a selective effect on

microbial growth. Xerphilic fungi have been found to grow at a low humidity 0.6% (Evans

and Niren, 1999).


2.5.4 NUTRIENTS
                                                                                          9

      Microorganisms depend on nutrients from meat product or meat for survival. Meat

contains protein, , fat vitamins and phosphorus which support the growth of microorganisms.

Pseudomonas aeruginosa synthesizes its vitamins and so cause spoilage even in a medium

without vitamin. Staphylococcus aureus require about 6.5% of sodium chloride for growth

and is usually found in salty meat products (Boles et al. 2000).




                                        CHAPTER THREE


                             3.0 MATERIALS AND METHODS


3.1    MATERIALS


3.1.1 CHEMICAL AND REAGENTS

      (1) Crystal violet


      (2) Iodine


      (3)Acetone (alcohol)


      (4)Safranin
                                 10

(5) Immersion oil

(6) Normal saline


(7) Hydrogen peroxide


(8) Distilled water


(9)Oxidase reagent


(10) Christensen’s urea broth


(11) Simon’s citrate agar


(12) Acetymythyl carbinol


(13) Buttered glucose broth


(14) Nepthol

(15) Sodium hydroxide


(16) Glucose phosphate peptone


(17) Methyl red


(18) Peptone water


(19) Tryptophan


(20) Kovac’s reagent


(21) Sugar solution
                                   11

    (22) Glucose

    (23) Sucrose


    (24) Lactose


    (25) Mannitol




3.1.2 GLASS WARES AND EQUIPMENTS

    (1) Cover slip


    (2) Glass slide

    (3) Rack


    (4) Glass rod


    (5) Filter paper


    (6) Bijou bottle


    (7) Wire loop


    (8) Incubator


    (9) Test tube
                                                                                         12

         (10) Durham tube

         (11) Autoclave


         (12) Bunsen burner


         (13) Weighing balance


         (14) Pasteur’s pipette


         (15) Sieve


         (16) Petri-dishes


         (17) Conical flasks


         (18) Mortar and pestle


3.2      SAMPLES COLLECTION

         Twenty skewers of suya meat were obtained randomly from suya vendors at popular

suya spots in Enugu. The samples were immediately wrapped in sterile aluminum foil to

prevent contamination and then transported to the laboratory for microbial analysis without

delay.


3.3      PRETREATMENT OF SAMPLES

         A suya piece from each sample was removed from the skewers, and mashed in a

sterile laboratory type mortar and pestle. 1g of the mashed suya meat was weighed and then
                                                                                            13

aseptically introduced into 9ml of sterile distilled water, properly shaken and sieved before a

twofold dilution was performed.


3.4 DETERMINATION OF TOTAL VIABLE COUNT AND                                  COLIFORM

COUNT

      A two -fold serial dilution was made for the suya meat samples in appropriate dilution

tubes. The media of choice are MacConkey agar and nutrient agar. The MacConkey agar is

a differential medium used in the differentiation of lactose fermenters though it grows on non

lactose fermenters. Nutrient agar is a supportive medium for the growth of most non-

fastidious microorganisms and also used to enrich media with blood serum. 1ml of each

dilution was pipetted and plated on nutrient agar and MacConkey agar using the spread

method. Incubation was       C for 24hours. Developed colonies were counted to obtain total

viable count and coliform counts respectively. Discrete colonies were purified by

subculturing into nutrient agar plates and were subsequently identified using standard

methods. (Bichanan and Gibbo, 1974).


3.5    PROCEDURE FOR CULTURING SAMPLES

      The samples were inoculated aseptically with a wire loop on the prepared. MacConkey

and Nutrient agar plates and incubated at 37 C between 18hours and 24hours. Then, the

plates were read for growth of organisms.




3.6   PROCEDURE FOR IDENTIFICATION OF THE ORGANISMS
                                                                                           14

      The isolates were characterized and identified based on their cultural characteristics

and biochemical reaction as follows:




3.6.1 GRAM REACTION

      This was carried out to differentiate gram position from gram-negative organisms.

Staphylococcus aureus and Escherichia coli were used as control organisms.




      METHOD:

     A wire loop was sterilized in Bunsen burner and allowed to cool then a loopful of

growth was collected from the agar plate and applied on a clean grease-free slide then a drop

of normal saline was added, emulsfied and heat fixed by passing over a flame three times.

The smear was flooded with crystal violet for 30-60seconds and then covered with iodine for

30-60seconds and then washed off; it was decolorized with acetone until no colour runs off

the slide and rinsed immediately. The slide was covered with safranin for 1minute and then

washed off with clean water. The slide was kept in a rack to air dry after wiping the back

with cotton wool.
                                                                                           15

        The stained smear was then examined microscopically under oil immersion at 100x

objective lens. Gram –positive bacteria appeared dark purple while gram-negative bacteria

appeared red.




3.6.2            MOTILITY TEST

        Motility test was aimed at identifying motile bacteria.


        METHODS

        A drop of normal saline was placed on a sterile slide and colony of test organism was

suspended and emulsified and then covered with a cover slip. The slide was examined

microscopically using 10x and 40x objective lens. Movement in different directions gave a

positive test.


        3.6.3 CATALASE TEST

        This was used to differentiate those bacteria that produce enzyme catalase such as

Staphylococcus aureus and Escherichia coli were used as positive and negative controls

respectively.


        METHOD

        Three milliliters (3ml) of hydrogen peroxide solution was poured into a sterile test

tube. Then a sterile glass rod was used to collect several colonies of the test organisms and
                                                                                          16

inoculate into the hydrogen peroxide solution. It was observed for immediate active bubbling

for positive test.




3.6.4    COAGULASE TEST

        This was used to identify Staphylococcus aureus which produces the coagulase

enzyme which cause plasma to clot by converting fibrinogen to fibrin. The slide method was

used.




METHOD

        A drop of sterile distilled water was placed on each end of a sterile slide. Then a

colony of the test organism was emulsified on each spot to make two thick suspensions. A

loopful of plasma was added to one of the suspensions and mixed gently. The slide was

examined for clumping or cloting of the organisms within 10seconds. Plasma was not added

to the second suspension which serves as control.


3.6.5 OXIDASE TEST

        This was carried out to identify bacterial species that will produce the

cytochromeoxidase enzyme. Pseudomonas aeruginosa and Escherichia coli were employed

as positive and negative controls respectively.
                                                                                               17




       METHOD:

       A piece of filer paper was placed in a clean Petri dish and 2-3 drops of fresh or nascent

oxidase reagent was added. A colony of test organism was collected using a glass rod and

smeared on the filter paper and observed. Blue-purple color within few a seconds showed a

positive test.


3.6.6 UREASE TEST

       This test was aimed at identifying Enterobacteria that produce urease enzyme, which

hydrolyze urea to give ammonia and carbon dioxide. Proteus and Salmonella were used as

control positively and negatively controls respectively.


       METHOD:

       The test organism was heavily inoculated onto Christensens urea broth in a bijou

bottle using a sterile wire loop and incubated at 35 C-        C for 18-24hours and examined,

thereafter a pink color in the medium showed positive test.


3.6.7 CITRATE TEST

       This test is based on the ability of an organism to use citrate as its source of carbon. It

was used to identify the Enterobacteria.


       METHOD:
                                                                                           18

      Simon’s citrate agar medium was prepared in a slant bijou bottle, then using a sterile

wire loop was used to inoculate the test organism onto the slant medium and incubated

at    C for 48hours after which it was examined for color formation. A bright blue color in

the medium gave a positive citrate test. Klebsiella pneumonia and Escherichia coli were

employed as positive and negative controls respectively.




3.6.8 VOGUES -PROSKAUER TEST


      This test was used to identify members of the Enterobactiaceae that produce

acetymythylcarbinol (acetone) a natural product formed from pyruvic acid in the course of

glucose fermentation.


      METHOD:


      Buffered glucose broth was inoculated with the test organism and incubated at        C

for 3days. Three milliliters (3ml) of nephtol was then added followed by 3ml of sodium

hydroxide solution, mixed well and allowed to stand for 1hour at room temperature. The

formation of a pink color in the medium within 1hour indicates a positive result. Klebsiella

pneumonia and Escherichia coli were used as positive and negative controls respectively.


3.6.9 INDOLE TEST

      This test was carried out for indole production by test organism which is important in

identifying enterobacteria.
                                                                                           19

METHOD:

A sterile wire loop was used to inoculate a colony of test organism into 2ml of peptone water

containing tryptophan. The tube was stoppered and incubated at 370C for 24hours. Kovac’s

reagent was added to the medium. Observation of red coloration on the surface layer within

10minutes showed a positive result.


3.6.10 CARBOHYDRATE FERMENTATION TEST

      This test is used to determine the ability of bacteria to utilize different sugars.

Examples are mannitol, glucose, lactose and sucrose.


      METHOD:

      The four sugar solutions were prepared and poured into test tubes well stopped with

Durham tube for gas collection. The sugar was autoclaved after which a loopful of test

organisms was introduced into the sugar solution (Buchana, and Caibbons. 1994). A change

in color from pink to yellow shows fermentation and collection of gas bubbles in the Durham

tube shows gas production which is a positive test. A control was set up without the

organism inoculated.


      3.6.11 METHYL RED TEST

      This was carried out to identify Enterobacteria based on the ability to produce and

maintain stable acid end product from glucose fermentation. Escherichia coli was used as

positive control.
                                                                                           20

      METHOD

      Glucose phosphate peptone water was used for inoculation of test organisms and

incubated for 48 hours at    c after which few drops of methyl red solution was added to the

culture and read immediately. Formation of red color immediately showed a positive test.
                                                                                            21

                                      CHAPTER FOUR


                                          4.0     RESULTS

      Suya samples collected randomly were carefully analyzed for their microbial profile.

The total viable count ranged from 1.9x         – 3.8x   . whereas total coliform count ranged

from 1.1x    -3.0x    as shown in Table 1. The characterization and identification result is

presented on Table 2. The isolates were identified as Staphylococcus (35%), Pseudomonas

(35%), Streptococcus (15%), and Escherichia coli (15%). The most frequently isolated

organism was Staphylococcus and Pseudomonas species.
                                                                    22

TABLE 1: TOTAL VIABLE AND COLIFORM COUNTS (103 CFU/G).


       S/N          MAcCONKEY AGAR         NUTRIENTAGAR

                       (coliform x103)   (total viable countx103)



         1                   1.1                   1.9

        2                    1.2                   2.0

        3                    1.3                   2.1

        4                    1.4                   2.2

        5                    1.5                   2.3

        6                    2.0                   2.4

        7                    2.3                   2.5

        8                    2.6                   3.2

        9                    2.9                   3.4

        10                   3.0                   3.8
                                                                                 26




TABLE 4: CHARACTERISATION/IDENTIFICATION OF ISOLATES

ISOL   GRAM CELLULAR IN C- V- M M O             U M C      LA GL S     PROBABL
ATE    REACT ARRANGE D T P R OT X               R AN A     C U U       E
       ION   MENT                                    T           C     ORGANIS
                                                                       M

S1     +VE    **         N   N   N   -   -  -   - D    +   +   +V A/ Staphyloc
                         R   R   R   V   VE V   V      V   V   E G occus
                                     E      E   E      E   E

S2     +VE    Cocci in   N   N   N   N   -  -   D NR -     N   N   N   Streptoco
              Chains     R   R   R   R   VE V        V     R   R   R   ccus
                                            E        E

S3     -VE    *          +   -   +   -   +V -   - +V   -   +   A/ D    E. coli
                         V   V   V   V   E  V   V E    V   V   G
                         E   E   E   E      E   E      E   E

S4     -VE    Rods       -   +   N   N   -  +   D -    +   -   D   -   Pseudom
                         V   V   R   R   VE V     VE   V   V       V   onas
                         E   E              E          E   E       E

S5     -VE    *          +   -   +   -   -  -   - +V   -   +   A/ D    E. coli
                         V   V   V   V   VE V   V E    V   V   G
                         E   E   E   E      E   E      E   E

S6     +VE    **         N   N   N   -   -  -   - D    +   +   +V A/ Staphyloc
                         R   R   R   V   VE V   V      V   V   E G occus
                                     E      E   E      E   E

S7     -VE    Rods       -   -   N   N   -  +   D -    +   -   D   -   Pseudom
                         V   V   R   R   VE V     VE   V   V       V   onas
                         E   E              E          E   E       E

S8     +VE    **         N   N   N   -   -  -   - D    +   +   +V A/ Staphyloc
                         R   R   R   V   VE V   V      V   V   E G occus
                                                                               27

                                   E      E   E      E   E

S9    -VE   Rods       +   +   N   N   -  +   D -    +   -   D   -   Pseudom
                       V   V   R   R   VE V     VE   V   V       V   onas
                       E   E              E          E   E       E

S10   +VE   Cocci in   -   N   N   N   -  -   D NR -     -   N   N   Streptoco
            Chains     V   R   R   R   VE V        V     V   R   R   ccus
                       E                  E        E     E

S11   -VE   *          -   -   -   -   -  -   - -    +   A/ D    -   E. coli
                       V   V   V   V   VE V   V VE   V   G       V
                       E   E   E   E      E   E      E           E

S12   +VE   **         N   N   N   N   -  -   - D    +   +   +V A/ Staphyloc
                       R   R   R   R   VE V   V      V   V   E G occus
                                          E   E      E   E

S13   +VE   Cocci in   N   N   N   -   -  D   - NR -     N   N   N   Streptoco
            Chains     R   R   R   V   VE     V    V     R   R   R   ccus
                                   E          E    E

S14   -VE   Rods       N   -   N   N   -  +   D -    +   -   D   -   Pseudom
                       R   V   R   R   VE V     VE   V   V       V   onas
                           E              E          E   E       E

S15   +VE   **         N   N   N   -   -  -   - D    -   +   +V A/ Staphyloc
                       R   R   R   V   VE V   V      V   V   E G occus
                                   E      E   E      E   E

S16   -VE   Rods       N   N   N   N   -  -   - D    -   +   +V A/ Pseudom
                       R   R   R   R   VE V   V      V   V   E G onas
                                          E   E      E   E

S17   -VE   Rods       -   +   -   -   -  -   D -    -   -   D   -   Pseudom
                       V   V   V   V   VE V     VE   V   V       V   onas
                       E   E   E   E      E          E   E       E

S18   -VE   *          +   -   +   -   -  -   - +V   -   +   A/ D    E. coli
                       V   V   V   V   VE V   V E    V   V   G
                                                                                    28

                              E   E    E   E       E   E     E   E

S19      +VE       Cocci in   N   N    N   -   -  D    - NR N    N   N   N   Streptoco
                   Chains     R   R    R   V   VE      V    R    R   R   R   ccus
                                           E           E

S20      +VE       **         N   N    N   -   -  -    - D   +   +   +V A/ Staphyloc
                              R   R    R   V   VE V    V     V   V   E G occus
                                           E      E    E     E   E




KEYS

      + positive


      - Negative


       NR Not relevant


       D Different strain given different result


       CT → Citrate test


       VP → Voges -Proskauer


       OX→ Oxidase


       CAT→ Catalase

       MOT→ Motility
                              29

  MR →Methyl red

  MAN → Mannitol


  SUC→ Sucrose


  LAC →Lactose


  A/G →Acid and gas


 GLU→Glucose


UR 一 Urease


 IND 乛 Indole


*一 rod in singles and pairs


**乛 Cocci in cluster
                                                          30




            TABLE 3 FREQUENCY OF OCCURRENCE OF ISOLATES


ISOLATE              FREQUENCY        PERCENTAGE%


Staphylococcus            6               35


Escherichia coli          4               16s


Pseudomonas               6               35


Streptococcus             4               15


Total                     20              100
                                                                                           31




                                     CHAPTER FIVE


5.0 DISCUSSION, SUMMARY, CONCLUSION AND RECOMMENDATIONS.


5.1 DISCUSSION

      Meat basically contains all the nutrients necessary for microbial growth and

metabolism, making it susceptible to microbial contamination. In view of the microbial

quality of meat and meat products, proper hygiene must be ascertained to ensure safety from

infection after consumption of such products and to promote quality assurance.


      In the present study, the microorganisms isolated were Staphylococcus species,

Escherichia coli and Pseudomonas species. The result was in consonance with the report of

Chukwura and Majekwu(2002) which stated that microbiological analysis of meat samples

in Awka urban of Anambra State, indicated contamination of meat samples with various

bacterial species including Staphylococcus aureus, and some enteric bacteria. Gilbert

Harrison(2001) also affirm that meat preserved with a certain amount of salt by so permit the
                                                                                            32

growth of Staphylococcus aureus whereas, the presence of some members of the family of

enterobacteriacea family is due to contamination from intestine of slaughtered animals.


        Four organisms were isolated from the suya sample in view of the of the unhygienic

condition of meat handling in Nigeria, the organisms isolated in this sturdy are the organisms

usually implicated in meat spoilage could always be suspected in connection with meat

contamination and spoilage.


        The presence of Staphylococcus species agrees with the report of cross contamination

from meat handlers during processing, since it is normal flora of the skin. ,(Gilbert and

Harisson 2001).Raw meat is usually carried on the body by butcher in Nigeria due to lack of

education Dada et al 1993,confirmed that coliform are introduced from the water used for

washing the meat which of course is always contaminated. This is also in agreement with the

report of Umoh (2004) that the also presence of Escherichia coli probably may arise from

the use of non –portable water during washing of raw meat


The meat also showed presence of Pseudomonas aeruginosa, which usually occurs in soil,

vegetation and surfaces of plants, humans and animals (Field, 2002).


        On the whole, the major sources of microbial contamination of suya meat appear to be

handling by butchers and the use of contaminated water and equipment. So control of suya

meat contamination can be achieved if aseptic techniques are employed during preparation of

suya.


5.2     SUMMARY AND CONCLUSION
                                                                                           33

      Suya meat constitutes a great source of protein which is needed for body building and

repair of worn out tissue in human. Improvement in the microbial quality of suya meat is

very important and adequate steps must be taken to prevent contamination and spoilage by

microorganisms.


The organisms isolated from the suya meat indicate that the standards of preparation and

preservation have not improved much over the years and facilities used for preparation are

not sterile. Aseptic techniques should be adequately employed in the meat industries so as to

reduce microbial load of meat and its products for safe consumption by consumers and thus

prevent food-borne diseases or infections.




5.3       RECOMMENDATIONS

            Quality control unit should be established in meat processing industries in

Nigeria and Hazard Analysis Critical Control Point (HACCP) concept should be applicable
                                                                                            34

to the processing and renderings of meat and suya meat beef products .this will go a long

way in reducing contamination and spoilage of meat products. Proper animal husbanding,

hygienic slaughter, adequate meat inspection, proper meat transportation sanitation of

utensils and equipment, portable drinking water and proper storage of meat should all be

employed to reduce microbial contamination. Research work should be earned out by

scientist in area of preservation of meat for a long time with shelf life of not less than one

year, a breakthrough will encourage a long period of storage, thus preventing contamination.




                     REFERENCES

Abdul, U.M., Beuchat, C.R., and Ammar, M. S. (1993). Survival and growth of Escherichia

    coli in ground roast beef as affected by pH, acidulates and temperature. Journal of

    Applied and Environmental Microbiology 59(8): 2364-2368.


Alexander,    J.W.,Jacob,     L.S.,   and    Nicholas,     B.N.    (1998).    Incidence     of

    enterobacteroria in meat processing. Journal of Food Science 27:177
                                                                                          35

Ayres, C.P. (1985). Microbiology of spoilt food and food stuffs. Journal of Food

    Microbiology 16: 206-212.


Boles, J. A., Rathgether, B.M. and Shand, P.J.(2000). Staphylococcus in salted meat product.

    Journal of Meat Science 55: 22-231


Buchanan, R.E. and Gibbons, N.E. (1994). Bergeys Manual of Determination Bacteriology.

    8th Edition: The Williams and wikins Co, Baltimore


Cannon, J.E., Morgan, J.B. and Mcketh, F.K. (1997). Meat contamination and poisoning.

    Journal of Muscle Food 7:29-36.


Chukwura, E.I. and Mojekwu, C.N. (2002). Prevalence of microbial contaminants of suya

    meat sold in Akwa Urban. Journal of Tropical Microbiology. 11:89-91.


Dineen, P., Emori, T.E. and Harley, R.N. (1999). Effects of Smoked Meat. Food

    Preservation Journal 69:25.


Evans, J.B. and Nicen A.T. (1999).Microbiology of Meat .In Bacteriology of Meat in the

    Science of Meat Production. Freeman Publisher, U.S.A


      Pp 276.


Favaretti, C. and Habida, J.(1999). Handling of meat. Journal of Food Processing and

    Preservation 12: 309-326.


Field, R.A. (2002). Enteric and food- borne illnesses. Advanced Food Research: 27:28-35
                                                                                        36

Forest, D.A., Harold, D.A., and Robert , A.M. (1975). Different Types of Meat and meat

    product consumed by Nigerians. In Principles of Meat Science. Public W.A. Freeman

    and Company, U.S.A. Pp. 4-178.


Fraiser C.W. Westholff C.D. (2001). Pathogens in meat and meat-borne illnesses. Food

    Microbiology.4th Edition . McCraw Hill Book Company, U.S.A. Pp. 401-411.


Gilbert, U. and Harrison, A. (2001). Occurrence of enterotoxin-producing . Staphylococcus

    aureus in meat market in Nigeria. Journal of Food Infection .56: 25-35.


Haman, D.O.(1977). Microbiology of Meat Food Technology 23(6):66-71


Igyor, M.A., Uma, E.N.(2005). Bacterial Quality of a smoked meat product (Suya). Nigeria

    Food Journal . 23: 233-242.


Jay, J.M. (2002). Suya in West African Recipes 12:15-20.


Judge, D.M., Robert, A .M. and Morris, M.J. (2002). Preparation of suya in Africa. Journal

    of African Foods. 20:52-55.


Lawries, R.A. (2001). Microbiology Growth in Meat. Meat Science 6th Edition. Pergoman

    Publishing Competition, Switzerland. Pp.43-49.


Lidway O.M.,Whyte, W., Lowe, D. (1996). Microbial Competition in meat. Journal of Dairy

    Science 70:822-826.
                                                                                       37

Nester, E.W., Aderson, D.G., Roberts, C.E., Pearsall, N.N. and Nester, M.T. (2001).

    Microbiology;A Human Perspective. Third Edition. McGraw Hill Company, U.S.A: Pp.

    822- 809.


Sokori, T.J. Anozie S.O. (1999). Meat spoilage . Journal of Food Production 53 (12):1069-

    1072.


Sokori, T.J. Anozie, S.O.(1999) . Journal of Tropical Microbiology. 7(2): 29-30.


Umoh, J.U. (2004). Critical Control Point of Beef Products and Food Resources, 22:80-85.


Walter, C. W. Kundin R.B. (2002).Faecal Contamination of Meat and Meat Products. Food

    Preservation Journal. 70: 88-92.
                                                                                          38




                                      APPENDIX 1


Preparation of Media and Reagents

The following media were used


Nutrient Agar (NA)


COMPOSITION


Content                                   Gram per Litre


 Lab. Lemco powder                             1.0

Yeast extract                                   2.0


Sodium chloride                                15.0


Agar                                           5.0


Peptone                                        5.0


pH                                              7.4


Twenty three grams of nutrient agar powder was transferred into one liter of distilled water

contained in a sterile conical flask. The mouth of the flask was plugged with non absorbent
                                                                                           39

cotton wool. Cover neck of flask with aluminum foil and tie firmly with a rope at the neck of

the flask. It was then mixed by shaking and brought to boil so as to dissolve completely and

autoclaved at       C for 15mins. It was allowed to cool at 45and mixed well before

dispensing aseptically in 20ml volume into Petri dishes. The medium was allowed to solidify

on these plates and were used thereafter.


MACCONKEY AGAR


 COMPOSITION


Contenet                             Gram per litre


 Pepton                                      20.0


 Lactose                                    10.0


 Bile salts                                 5.0


 Sodium chloride                            5.0


 Neutral red                                0.075


  Agar                                      12.0


  Distilled water                           100ml


  PH                                        7.6


PREPARATION
                                                                                           40

       Twenty eight grams of MacConkey agar powder was weighed and transferred into

one liter of distilled water contained in a sterile conical flask. The mouth of the flask was

plugged with non absorbent cotton wool. Cover neck of flask with aluminum foil and tie

firmly with a rope at the neck of the flask. It was then mixed by shaking and brought to boil

so as to dissolve completely and then autoclaved at           C for 15mins. It was allowed to

cool to 45   and mixed well before dispensing aseptically in 20ml volume into Petri dishes.

Prior to incubation, the surface of the agar was dried by partial exposure at 37C. The

appearance of the plate was clear pink/red. The media was preserved in the fridge. Nutrient

agar is used as a concentration of 2.8g in every 100ml of distilled water.


Christensens urea broth

Urea broth base                                     95ml

Sterile urea solution40% w/v                        5ml


Christensens is used test if an organism is positive or negative using bacteria species.


Simon Citrate Agar (SCA)

This medium was used for the differentiation of Enterobacteriaceae based on the utilization

of citrate as the sole source of carbon.




                                       COMPOSITION

Magnesium sulphate                                     0.2g
                                                                                          41

Sodium ammonium sulphate                              0.8g

Ammonium dihydrogen sulphate                          0.2g


Sodium citrate tribasic                               2.9g


Sodium chloride                                        5.0g


Bromothy molblue                                      0.08g


Agar                                                  15g


Distilled water                                       1000ml


pH                                                    6.9


PREPERATION


       The powered urea agar was used and was prepared as directed by the manufacturer.

2.4g of urea agar was suspended in 95ml of distilled water and was dissolved by boiling. The

medium was sterilized by autoclaving at 115 C for 20minutes. The medium was cooled to

about 50 C and 40% w/v sterile urea solution was added. The medium was dispensed into

bijou bottle and were allowed to solidify in slant position. They were therefore used.


                                        APPENDIX 2


 Preparation of reagents.


(1)    Methyl red solution

To make 50ml
                                                                                           42

      Methyl red (pH indicator)                             0.05g

      Ethanol (ethyl alcohol) absolute                      28ml

      Distilled water                                       22ml

      (a)   The methyl red was weighed on a piece of paper (pre weighed) dissolved in ethanol

      and water.

      (b)   It was transferred to a clean brown bottle and the bottle was labeled.

      (c)   It was stored at room temperature in a dark place.

      (2)   Crystal violet gram stain

      To make 1litre

      Crystal violet                                         20g

      Ammonium chloride                                       9g

      Ethanol or methanol absolute                           95ml

      Distilled water                                        1litre




(3)   Peptone water

      To make 65bottle

      Peptone                                                2g

      Sodium chloride                                        1g

      Distilled water                                        200ml

      pH                                                     7.6
                                                                                     43

(a)   The peptone water and salt was dissolved and dispense in 3ml amounts in screw cap

bottles (Bijou bottle).

(b)   It is sterilized by autoclaving (with caps loosened) at 121 c for 15 minutes and

allowed to cool with the cap tighten and the bottle was labeled.

(c)   It was stored in a cool dark place.

Oxidase Reagent

To make 10ml:

Tetramethyl-p- phenylenediamine                             0.1g

Dihydrochloride

Distilled water                                             10ml


The chemical was dissolved in distilled water and was used immediately.




Acetone–alcohol decolorizer:

  To make 1 litre:


  Acetone                                                    500ml


Ethanol or methanol, absolute                                475ml


Distilled water                                              25ml
                                                                                         44

The distilled water was mixed with ethanol and was transferred into a screw cap bottle of 1

litre capacity. The acetone was measured and added immediately to the alcohol solution and

then mixed well.

The bottle was labeled and indicated highly inflammable then store in a safe place at room

temperature.


Glucose phosphate peptone water:

   To make about 50 bottles :


Peptone                                                0.5g


Glucose                                                 0.5g


di-potassium hydrogen phosphate                        0.5g


Distilled water                                       100ml


The peptone and phosphate salt were dissolved in water by steaming, and allowed to cool,

filtered and the pH was adjusted to 7.5.

The glucose was added, mixed well and dispensed in 2ml amounts in small screw -cap tubes

or bottles. It was sterilize by autoclaving (with cap loosened) at 11 C for 10minutes and

allowed to cool, then tighten the container tops and label.


(c) Store in a cool dark place or as 2-8 C.
45

				
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