1

BACTERIOLOGICAL EXAMINATION OF MEAT

PIE SOLD WITHIN THE ENVIRONMENT OF
NNAMDI AZIKIWE UNIVERSITY ENVIRONMENT
AWKA
ABSTRACT
A total of eight (8) meat pies samples purchased from four (4) different
standard eateries in Nnamdi Azikiwe University, Awka, Anambra state, were
analysed for presence of pathogenic bacteria using standard microbiological
techniques. The following bacterial genera were isolated and identified from the
meat pie, Staphylococcus (42.9%), Escherichia (42.9%), and Bacillus (14.3%). From
the frequency of occurrence it was observed that Staphylococcus and Escherichia
spp predominantly occurred in all the samples. Also, there were equal occurrence of
gram positive and gram negative bacteria (in the ratio 2:1) in the entire sample
which gave rise to 50% and 50% occurrence respectively.

TABLE OF CONTENTS

Title page i

Approval ii

Certification iii

Dedication iv

Acknowledgement v

Table of content vi

Abstract viii

Introduction 1
 2

Literature Review 5

Pathogen Associated With Meat Pie 5

Health Implications of Contaminated Meat Pie 7

Staphylococcal Food Poisoning/Intoxication 7

Infection due to Escherichia Coli 8

Infection Due To Clostridium Perfrigens 11

General Prevention and Control of Food Measures Hazards 13

Previous Studies and Investigations 14

Materials and Methods 15

Study area 15

Sample Collection 15

Sample Analysis 15

Processing of the Samples 16

Isolation of Pure Culture 17

Identification of the Isolates 18

Morphological Examination Gram 18

Biochemical Test 20
 3

Results 24

Discussion 26

Conclusion 28

References 29

Appendix 1 32

Appendix 2 34

Appendix 3 35

INTRODUCTION

A meat pie is a savoury pie with a filling of meat and other savoury

ingredients. Principally popular in Europe, Australia, New Zealand and South

Africa. Meat pie differ in pastry in the sense that a pastry is typically a more

portable on the go items, as opposed to more convectional pie

(www.wikipedia.org/wiki/meat-pie, accessed 5/2/2012). Meat pie is one of the

meat popular snacks in Nigeria; the best Nigerian meat pie is moist, yummy

having a filling of minced meat, potatoes and carrot. Vegetable such as green

pepper may be added as a matter of choice.

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A general characteristics of our society showing a social pattern characterised

by increased mobility large members of itinerary workers and less family or

home centred activities. This situation however has resulted in meat pie taken

outside home. These food vendor services has become on the increase and

responsibility for good manufacturing practises of food such as sanitory

measures and proper good handling has been transferred from

individual/families to the food vendors who rarely enforce such practises (Musa

et al., 2002).

Food borne diseases are diseases resulting from ingestion of bacteria, toxins and

cells produced by microorganism present in food. Food borne illness is a

major international health problem with a consequent economic reduction. It

has been estimated that seven pathogens are associated beef product (meat pie)

which include Escherichia coli, Salmonella spp, Listeria monocytogens

Campypylobacterjejuni, Clostridium perfrigens, Toxoplasma gondii,

Staphylococcus aureus accessed for approximately 3.3-12 million cases as

food borne illness (Taken et al; 1996; Buzbyet al., 1997). Important bacteria

food borne pathogen may be implicated either of the two primary type of

food released diseases: food borne ingestion and food intoxications.

 5

A food borne ingestion involves the ingestion of pathogen, followed by growth

in the host, including invasion and or the release of toxin (Prescott et al., 2008).

Some of the major diseases of this type associated with beef product include

Salmonellosis, listeriosis and hemorrhagic colitis caused by a srain of

Escherichia coli known as EnterohemorrhagicEscherichia coli (Dolobeset al.,

2011).

Food intoxication produces symptoms after food is consumed because of the

growth of the disease causing microorganisms (bacteria) is not required (Prescott

et al, 2008). Major diseases of this type associated with meat pie include

botulism and staphylococcal food intoxication. Food supply issues of processed

meat is usually as a result of contamination (bacterial) introduced exogenously

during activities such as harvesting, processing and preparation (Toroket al.,

1997) or endogenous contamination due to the presence of bacterial

pathogens inherent in the cattle and such serve as major reservoir and vehicle

for meat of these pathogens.

The student population usually consumes meat pie in large numbers. Issues

of gastrointestinal disturbances are usually associated with consumption of such

product when they are prepared, handled or served in unhygienic ways. Meat

pie often the snack of choice in most social gathering. When such products are
 6

prepared far inadvance of service and held in warm temperature (temperature

abuse) multiplication of some bacterial and growth of their toxins is encouraged

and tends to increase.

In most countries the most common food borne illness is Staphylococcus

food intoxication. Enterotoxigenic Staphylococcus strains and E. coli strains

have been so latent from food implicated to illness. E. coli and S. aureus are

named flora in humans and animals. Their presence in food is an indication of

excessive human handling. They produce disease when the bacterial contaminate

the food. They produce some enzymes which are very harmful and extra cellular

substances some of which are heat stable enterotoxin that render food dangerous

even though it appears normal (Prescott et al., 2008).

Once the bacteria has produced toxin, the food can be extensively and

properly cooked killing the bacterial without destroying their toxin. Many of the

toxin are gene based that is carried on plasmid. The intensity of the sign and

symptoms may vary with amount of contaminated food ingested and

susceptibility of individual to toxins. Escherichia coli is commonly used as a

surrogate indicator. Its presences in food (meat pie) generally indicate direct or

indirect food combination. However in Nigeria, a number of foods has been

reported to have high incidence of bacteria but their limited information on the
 7

health challenges from food borne diseases from meat pie retailed within a

highly populous community.

The objective of this study is focused on assessing the bacteriological state of

meat pie sold in standard eateries in Nnamdi Azikiwe University, Awka,

characterising and identifying the bacterial isolates and to suggest possible

ways of prevention and controls of the occurrence of these pathogenic bacteria

in meat pie.
 8

LITERATURE REVIEW Major

Pathogen Associated With Meat Pie

Bacteria can be found in many foods including meat pie, if the food is not

prepared properly. Bacteria in food cause ingestion, especially when numerous.

Bacteria did not stop growing after it have been eaten and might even further

grow in intestines causing illness. This is called food intoxication, which is

caused by consumption of toxins produced in food by bacterial growth. It is not

the bacteria itself that causes illness but the toxin, which does not after the

appearance, odour of flavour of the food. Major pathogen associate with meat

pie are salmonella sp, Escherichia coli, Listeria monocytogenes, Campylobacter

jejuni, Closridiumperfrigens, Toxoplamagondii and Staphylococcus aureus.

Salmonella

Once eaten the food infected by Salmonella bacteria it causes

Salmonellosis. Salmonella will continue to grow in the intestine after it is

ingested, setting up an infection and causing illness. The severity of the illness

depends on the size of the dose, the resistance of the host and the specific

strain of Salmonella. It is spread through indirect and direct contact with

intestinal content or excrement of animals, including humans often by hands that

are not washed after using the toilet.

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Escherichia coli

Escherichia coli belong to the family of microorganisms called coliforms. Most

times Escherichia coli are helpful to humans living in the intestine and

preventing the growth of more harmful pathogen. One strain, however, often

cause a deadly disease canned beef is most commonly associated with

Escherichia coli but other foods such as raw milk, unpasteurized apple juice

and cider, sprout, lettuce and spinach have been also implicated. It is prevented

by thorough washing and cooking of the raw product and by avoiding

recontamination of cooked meat with raw meat.

Cattle present a major reservoir of Escherichia coli 0157:117 (Whippet al., 1994)

and while present at low frequencies pose significant hazards in terms of human

disease. This means that a significant number of animals will carry these and

other highly undesirable human pathogens into abattoir environment and

processes, leading to direct and indirect contamination of equipment and

exposed meat surface during slaughter and dressing activities (Gill, 1998).

Environmental conditions that affect the growth of bacteria can include storage

temperature, moisture availability and physical state of the meat pie. Spoilage

bacteria that find condition most favourable will begin to grow even at

refrigerated temperature.
 10

Health Implications of Contaminated Meat Pie

Staphylococcal Food Poisoning/Intoxication

Staphylococcus aureus is a gram positive coccus resistant to drying and

radiation, found in nasal passage and on skin of humans and other mammals

worldwide. From these sources, it can readily enter food. If the bacterial is

allowed to incubate in meat pie, it produces heat stable enterotoxins that render

the food dangerous even if it appears normal. Once the bacterial have toxin,

the food can be extensively and properly cooked, killing the bacterial without

destroying the toxin (Prescott et al.,2008).

Thirteen (13) different enterotoxin have been identified; enterotoxin A, B, C1,

C2, D and E are most common (Enterotoxin A and B are super antigen) the

intensity of the signs and symptoms may vary with the amount of contaminated

food ingested and susceptibility of the individual to toxin. Typical symptoms

include severe abdominal pain, cramps, diarrhoea, vomiting and nausea. The

onset of the symptoms is rapid (usually 1-8 hours) and of short duration

(usually less than 24 hours). The intensity rate of staphylococcal food

poisoning is negligible among healthy individuals. Diagnosis is based on

symptoms or laboratory identification of bacteria from foods by animal toxicity

test or an antibody based methods. Treatment is with fluid electrolyte replacement

 11

prevention and control of personal responsible for food preparation and

distribution (Prescott et al., 2008).

Infection due to Escherichia Coli

Escherichia coli commonly abbreviated as E. coli is named after (Theodore

Escherich) is a gram negative, facultatively anaerobe rod shaped, non-spore-

forming bacterium. It can live in a wide range of substrate. It is commonly found

in the lower intestine of warm blooded organism (endotherms). Most E. coli

strains are harmless but some such as 0157 :H7 are occasionally responsible for

recalls. Optimal growth of E. coli occurs at 37oC (98.6F) but some laboratory

strains can multiply at temperature of up to 49 o C (120.2F). E. coli is a

member of the Escherichia family, enterobacteriaceae. Contaminated food

and water are the major source by which the bacteria are stored. Food poisoning

caused by E. coli is usually caused by eating unwashed vegetables or

undercooked meat. Selected trains can cause a wide variety of infection in

hospitals and community settings. These include gastroenteritis, urinary

infection and inflammatory dysentery (Splanger, 1992)

Certain strain of E. Coli such as 0157:H70121 & 0104:H21, produce potentially

lethal toxin. E. coli is notorious for causing serious and even life threatening

complications such as haemolytic-uremic syndrome. E. coli habour both heat

 12

stable and heat labile enterotoxin. A subgroup called enterohaemorrhagic E. coli

(EHEC) can cause severe potentially fatal illness known as haemorrhagiccolitis

which is characterised by bloody diarrhoea and severe abdominal pain (Dolobes

and Doyle, 2001)

Transmission of pathogenic E. coli often occurs via faecal-oral route. Common

causes of transmission include unhygienic food preparation, farm

contamination due to manure, fertilisation, and irrigation of crops in

contaminated grey water or raw sewage, direct consumption of sewage water.

Dairy and beef cattle are primary the reservoir of E. coli 0157 :H7 and they can

carry it asymptomatically and shed it in their faeces. Food products associated in

E. coli outbreaks include new ground beef, raw milk, sausages, meat pie and

hamburger. According to the United States Food and Drug Administration, the

faecal-oral cycle of transmission can be disrupted by cooking food properly,

preventing cross contamination involving barriers such as gloves for food

workers and including health care policies.

Salmonellosis

Salmonella is a genus of rod shaped, gram positive, non-spore forming

andpredominantly motile enderic bacteria with diameter around 0.7-1 .5um,

 13

length from 2-1 5um and flagella which grade in all direction (peritrichous). They

cause illness like typhoid fever, paratyphoid and salmonellosis. The initial

source of the bacterium is the intestinal tract of birds and other animals.

Human acquire the bacterial from contaminated food such as beef product

(meat pie), egg, egg product or water.

Once the bacterium is in the body the incubation time is only about 8-48 hours.

The disease results from a true food-borne infection because the bacterial

multiply and invade the intestinal mucosa, where they multiply and invade

intestinal mucosa, where they produce an enterotoxin and a cytotoxin that

destroy the epithelial cell. Abdominal pain, cramps, diarrhoea, nausea,

vomiting, and fever are the most prominent symptoms, which usually passed

for 2-5 days but can last for several weeks. During the acute phrase of the

disease, as many as one billion Salmonella can be found per gram of faces.

Laboratory diagnosis is by isolation of the bacterium from food or from patient’s

stool. Treatment is with fluid and electrolyte replacement. Preventions depend

on good processing practises, proper refrigeration and adequate cooking (Prescott

et al, 2008).
 14

Infection Due To Clostridium Perfrigens

Clostridium perfrigens formerly known as Clostridium welchii is a gram positive

rod shaped anaerobic spore forming bacterium of the genus clostridium.

Clostridium perfrigens is ever present in nature and can be found as normal

component of decaying vegetation, marine sediment, the intestinal tract of

humans and other tines can be ingested and not cause any harm (Prescott et al.,

2008).

Infection due to clostridium perfrigens show evidence of tissue necrosis,

bacteraemia, emphysematous cholecystitis and gas gangrene, which is also

known as clostridialmyonecrosis. In the united states and united kingdom,

clostridium perfrigensbacteria are the third most common cause of food borne

illness with poorly prepared meat andpoultry as the main culprit in harbouring

the bacterium. The clostridium perfrigensenterotoxin (CPE) mediating the

disease is heat labile (dies at 74oC) and can be detected in contaminated food if

not properly heated. Incubation time is between 6-24 (commonly 10-12) hours

after ingestion of contaminated food. Often, meat is well prepared but too far in

advance of human consumption since clostridium perfrigensform spores that can

stand cooking temperatures is allowed to stand for long enough germination

occurs and infection bacterial colonies develop. Symptoms typically include

 15

abdominal clamping and diarrhoea, vomiting and fever unusual. The whole

course usually resolves within 24 hours.

In blood agar plates, clostridium perfrigensgrown anaerobically produces beta-

haemolytic flat spreading, rough translucent colonies with

iregularmargins(Prescott et al., 2008)

Listeriosis

Listeria monocytogenes is a common gram positive rod that can be isolated from

soil vegetation and many animal reservoirs. Recent evidence suggests that a

substantial number of cases of human listerosis are attributed to the food borne

transmission of listeria monocytogenes. Listeria out breaks have been traced to

sources such as contaminated milk, soft cheese, vegetable and meat. Unlike

many of the food borne pathogens which cause primarily gastrointestinal illness,

listeria monoctogenes cause inclusive syndrome such as meningitis, sepsis, and

still birth. (Prescott et at., 2008).

 16

Diagnosis of ListeriosisIs By Culture of Bacterial Treatment is by intravenous

administration of either ampicillin or penicillin because listeria monoctogenes

is frequently isolated from food, the USDA (US Department of Agriculture)

and manufactures are pushing measures to reduce the contamination of food

product by this bacterium.

General Prevention and Control of Food Measures Hazards

The critical control point (CCP) according to a study published in journal of

food safety in 2004 in a point step procedure in a food process at which control

can be applied and as a result, food hazard can be prevented, eliminated or

reduced to acceptable level. Food processors must use food CCP limits that

have been scientifically validated to prevent growth of pathogens (government

should provide basic amenities such as steady power supply and access to

portable water. Education of handlers is equally important. Above all high

hygiene should be maintained in all food production sectors (Anon, 2002).

 17

Previous Studies and Investigations

Data on issue of food borne diseases are well documented worldwide. Food

borne illness is a major international health problem with consequent economic

reduction. Foods borne disease are disease resulting from ingestion of bacteria,

toxin and cells produced by microorganisms (Doyle et al., 1999).Outbreaks of

food borne diseases are caused by food that are contaminated intrinsically or

that become contaminated during harvesting, processing or preparation

(extrinsically) (Toroket al., 1997).

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MATERIALS AND METHODS

All materials used in the experiment are presented in the appendix

Study area

The study area is Nnamdi Azikiwe University, Awka, Anambra state. Samples

were purchased from standard eateries including- Science village, Madonna,

School gate and Coke centre. These study area were chosen because of their

location and large volume of patronage per day.

Sample Collection

Four samples of fresh meat pie were purchased from four (4) different

standard eateries (one from each) as listed in the above section (study area).

These samples were aseptically collected in a clean polyethylene bag and

immediately transferred to the laboratory for further analysis.

Sample Analysis

Sample analyses of the meat pie were carried out in two (2) parts:

a. pastry b. minced meat. This is because of the difference in mode

of preparation of the pastry/minced meat before the final meat pie is prepared.
 19

Processing of the Samples

Ten (10) grams of each food sample from each of meat pie from a particular

eatery was aseptically homogenised in a sterile beaker containing 200ml of

sterile water. This was repeatedly done for all the four eateries. Cultures were

incubated at 37OC for 18-24 hours.

A 10-fold dilution of all the homogenate were carried out using sterile distilled

water as the diluents. Five sterile test tubes containing 9ml sterile distilled water

each were arranged in a test tube rack (for each of the four homogenous

samples contained in the beakers). One millilitre (1ml) of each homogenous

sample was serially diluted in the test tubes containing the 9ml sterile distilled

water each using 10ml volume glass pipette, each transfer followed by a gentle

agitation in other to mix the contents uniformly. The procedure was repeated

for all the homogenate samples. The serial dilutions were done aseptically

beside lit Bunsen burner to avoid contamination.

 20

Culture

The serially diluted samples were used to inoculate already prepared and

poured nutrient agar medium. Each dilution was inoculated using the pour plate

method as described by Singleton (1999).

0. 1ml aliquots of 10-1 dilution of the homogenate were inoculated using pour

plate method mixed well and inoculated plates were inverted and incubated for

24 hours at 370C.

Plate count

The culture plates were inspected after 24 hours incubation. About 30 to 275

colonies forming unit (CPUs) per ml was observed.

Isolation of Pure Culture

Isolation of pure culture was done by randomly sub culturing various colonies

from the spread plates unto prepared Nutrient agar by employing streak method

as described by Cheesbrough (2002). A colony from the original culture plate

was introduced at one end of a new plate (taking care not to touch the edge of

the plate to avoid contamination). Using a sterile wire loop the inoculum was

streaked across the agar in four different directions with the last streak (tail)
 21

being done in zig zag manner. The Nutrient agar plates that contain the discrete

colonies were incubated at 37oC for 24 hours.

Identification of the Isolates

The discrete (pure) colonies were then isolated and identified based on

colony morphology as well as biochemical test.

The following test were carried out as described as by Cheesbrough (2003)

and Ogeleke and Manga (2008).

Morphological Examination Gram

Staining

Gram staining is the basic test in determinative bacteriology used to group

bacterial into gram positive and gram negative organisms based on their different

reactions to gram’s stain.

According to (Nester, et al, 2006) the following stepwise procedure was carried

out during gram staining.

A thin smear of the bacterial isolate from a 24-hour plate was made on a clean

slide. This was done by dropping a loopful normal saline on the slide and a very

minute quantity of the test isolate was emulsified with the normal saline to air
 22

dry, then it was heat fixed over a Bunsen burner flame by passing the slide in

east-west direction twice. The smear was flooded with 0.5% crystal violet

(primary stain) for 60seconds and then rinsed off with distilled water. It was

then flooded with Lugol’s iodine and allowed for 1 minute. Again, it was

washed with water. It was then decolorized with acetone iodine for 5 seconds

and rinsed off immediately. Finally, the smear was flooded with safranin

(counter stain) for 1 minute then rinsed off with distilled water. The stained

smear was allowed to air dry and then viewed under light microscope using

x100 objective lens with a drop of immersion oil.

Spore Test

This test was basically done to know if the organisms were spore former or non-

spore former.

Bacterial film was made on a clean slide dried and heat fixed with minimal

flaming. The slide was placed over the rim of beaker of boiling water with the

bacterial film uppermost. The film was flooded with 0.05% aqueous solution of

malachite green. When large droplets have condensed on the underside of the

slide and left to act for one minute while the water continue to boil, the slide

was then washed with cold water, treated with 0.5% safranin solution and left for

30seconds. The slide was then washed, dried and viewed under microscope using
 23

x40 objective lens. This method coloured the spore green and vegetative bacilli

red.

Motility Test

Tubes containing sterile nutrient agar are allowed to semi-solidify (molten)

slanting the tube. The medium is then inoculated by plugging a long straight

wire charged with the test isolate vertically into the centre of the tube. Motility

is indicated by growth of the organism away from the vertical line.

Biochemical Test

Coagulase Test

This test was performed to detect the presence of an enzyme coagulase

which converts a soluble plasma protein, fibrinogen to insoluble fibrin (Clot).

Slide method was used in performing the test. A drop of water was placed on a

grease free slide and a loopful of the isolate and gently mixed, then a drop of

plasma was placed on the mixture and observed. The presence of agglutination

or clumping indicates a positive result, while negative result was indicated by

absence of agglutination.
 24

Catalase Test

Catalase is an iron-containing enzyme which catalyses the decomposition of

hydrogen peroxide (H2O2) to water and oxygen.

The slide technique as described by singleton (2002) was used. A dense

inoculum of a 24 hour culture of the isolate was placed on a clean grease free

slide using a sterile wire loop. Then, a drop of hydrogen peroxide (H2O2) was

placed beside the dense inoculum and mixed. Catalase positive organisms

produced effervescence reaction immediately unlike the catalase negative that

showed no visible reaction.

Citrate Test

Test isolate was inoculated into test tubes containing sterile Simmon’s citrate

agar sand then left on the laboratory work bench for 24 hours. Appearance of a

blue colouron the surface of the agar indicates a positive result while the

negative isolate remained the same as indicated by all control setup.

Indole Test

The test organism was inoculated in test tubes containing 3ml of sterile peptone

water. It was left on the laboratory work bench for up to 48 hours. 0.05ml of
 25

Kovac ’s reagent was added and shaken gently. Appearance of a red colour in

the surface layer within 10 minutes indicates a positive result.

Methyl Red Test

The test organism was incubated onto sterile glucose phosphate peptone water

and left on the laboratory work bench for 48 hours. The five drops of methyl

reagents were added, mixed and the result read. The production of bright red

and yellow colour indicated a positive and negative result respectively.

Sugar Fermentation Test

This test was performed to detect the ability of isolate to utilise various sugars

with

the production of acid and gas or acid only. The following sugars, sucrose,

glucose, lactose, and were tested using bromothymol blue as indicator and

Durhamtubes to detect gas production. The stepwise procedure was described by

Nester et al., (2004).

1.5ml of peptone water was prepared to 100ml of water according to the

manufacturer’s specification and 10ml bromothymol with 0.5g of sugar, this

was done for each of the sugars( glucose, lactose and sucrose), It was then
 26

dispensed in 10ml portion into test tubes. Carefully, clean Durham tubes were

inserted into the test tube and was labelled accordingly. This whole

preparations was then sterilised at 115oC for 10 minutes.

The test isolate (24 hours culture) were inoculated into each already labeled test

tubes containing sugar basal medium, indicator and Durham tube and then left

on the laboratory work bench for 24-48 hours.

Positive results were indicated by colour change of medium from blue to yellow,

thus, indicating acid production from sugar metabolism, while gas

production was indicated by bubble or air space formation in Durham tubes.

The control tubes which contain sterilised peptone water, indicator and sugar

but without the test isolates remain ever the same and then used to make

comparison and pin-point positive and negative result.

 27

RESULTS

A total number of three (3) bacteria isolates were recovered from the four(4)

fresh meat pie samples. These include Staphylococcus spp 3 (42.9%), Escherichia

spp3 (42.9%), and Bacillius (14.3%). The frequency of the isolation as shown in

table 2.

Table: Morphological and Biochemical characteristics of Bacteria Isolates

Methyl red test

Coagulase test
Nutrient agar

Catalase test
Gram stain

Citrate test

Indole test
Motility

Glucose
Isolates

Sucrose
Lactose
Shape

Spore

Staphylococcus Smooth + Coccus - - + + - - - A A A

spp pink
colonies
Escherichia Red - Rod - - + - + + + A/G A A
spp colonies
Bacillus spp Pale + Rod + + + - + - - A - A
pink
colonies

Total

Keys

+ = Positive - + Negative

A/G = acid production and gas production

A = acid production only

Table 2 :Frequency Distribution of the Bacterial Isolates

 28

Isolates Frequency Percentage occurrence (%)

Staphylococcus spp 3 42.9

Escherichia spp 3 42.9

Bacillus spp 1 14.3

Total 7 100
 29

DISCUSSION

In this study, three (3) genera of bacteria were isolated and identified from

the meat pie. These include Staphylococcus, Escherichia, and Bacillus.

These bacterial contaminations are known to be a major course of food

borne diseases giving rise to acute to chronic illness as reported by

Edema et al., (2005).

From the frequency occurrence the predominant organisms are Staphylococcus

spp, Escherichia spp. The presence of these microorganisms in processed

ready to eat meat pie depicts a deplorable state of poor hygiene and sanitary

practises employed in processing and packaging of these beef products. This

findings is in consonance with the report of Okonkoet al., (2009)

From the results obtained, the meat pie was contaminated with high level

of Staphylococcus aureus and Escherichia coli. These agrees to previous

reports that foods of animal origin either cooked or uncooked were

predominatly contaminated with E. Coli and Staphylococcus. (Waites and

Arbothnot, 1999) reported of Staphylococcus aureus and E. Coli

contamination in minced beef, sausage rolls and pies.

 30

They reported 60% prevalence of Staphylococcus aureus, 50% E coli, 40%

Shigella, and 30% Morganellamorgani. From recent findings food mixtures

such as pastries, sauces, soups, salads etc have been frequently

incriminated in food poisoning outbreaks. The isolated Bacillus species

have been incriminated to contribute towards the life threatening illnesses.

This still boils down to contamination in food handling hygiene technique,

starting from processing raw material to the finished products. According to

Edema et al., (2001) and Okonkwoet al., (2008), the presence of E.coli is an

indicator of faecal contamination of water sources that is utilised in processing

these meat products.

From the results, it is clear that the meat pie had a lot of bacterial

contamination. This may be because beef offers a rich material media for

microbial growth (Phillips, 2003). These contaminations could also be as a

result of excessive unhygienic handling, during preparation.

 31

CONCLUSION

Various bacteria genera were isolated and identified from the samples,

These include, Staphylococcus, Escherichia, and Bacillus. The result revealed

that the samples (meat pie) sold in these eateries in Nnamdi Azikiwe

University, Awka were directly or indirectly contaminated with high levels

of pathogenic bacteria, most especially staphylococcus spp and Escherichia

spp. This study clearly confirmed the deplorable state of ready to eat food

(meat pie). However occurrence of the pathogens can be essentially

reduced or prevented by employing good manufacturing practises

(GMP).

From this research the issue of food safety is of paramount importance. Thus to

safeguard against the risk of food poisoning, there is need to educate and

advocate for good manufacturing practises (GMP) among beef processors.

Also relevant agencies in Nigeria such as National Agency for Food

and Drug administration and Control (NAFDAC) and Standards

Organisation of Nigeria (SON) need to ensure and enforce strict compliance to

hazard analysis critical control point (HACCP) in all food production sectors

in Nigeria. The government can equally contribute by provision of steady

power supply and access to portable water.

 32

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 35

APPENDIX 1

Experiment and Apparatus

Aluminium foil
Autoclave

Beaker

Bijoux bottles
Centrifuge

Conical flask Cotton wool

Durham tubes Hot air
oven Measuring cylinder

Microscope

Masking tape Petri

dishes

Pipettes
 36

Refrigerator

Slides

Spatula

Test tubes

Test tube rack Weighing balance Wire loop

Sugars

 Glucose

 Sucrose

 Lactose

 Maltose
 37

Appendix 2

Reagents

 Acetone-alcohol decolouriser

 Crystal violet stain

 Ethanol (70%)

 Lugol’s iodine solution

 Safranin

 Normal saline (0.85%)

 38

Appendix 3 Media
Composition and Preparation

Nutrient Agar Medium

This is a general purpose bacteriological medium capable of supporting the

growth of most organisms owning to the its nutrient composition. It was used to

enumerate the variable colonies of bacterial in the samples and to store organism

in bijoux bottles for reference purposes.

Composition

Lab-lemco powder 1g/l

Yeast extracts 2g/l

Sodium chloride 5g/l

Agar powder 1 5g/l

pH 7.4

Peptone 5g/l

Preparation

This medium was prepared as directed by the manufacturer. 7gram of nutrient

agar was weighed out using the weighing balance. 250ml of water was measured

out using measuring cylinder, from this volume, 100ml was used to soak and

dissolve the nutrient agar powder then the volume was brought up to 250ml in

a conical flask, then it was heated to properly dissolve the agar. This
 39

suspension in a conical flask clogged and soaked with cotton wool and

aluminium foil respectively, was sterilized in the autoclave at 121oC for 15

minutes. The molten medium was allowed to cool to 45oC before being poured

into sterile Petri dishes in which was allowed to set. the slant was prepared

pouring the medium molten agar into bijoux bottle and all owing it to set in a

slant position.

Peptone Water Medium

This basal medium was used to grow the isolates for motility and sugar

fermentation test. It is a general purpose medium

Composition

Peptone 10g/l

Sodium chloride 5g/l

pH 7.6

Preparation

It was prepared as instructed by the manufacturer by dissolving 7.5g of the

peptone water in 500ml of water. This was sterilised in the autoclave at 121oC

for 15 minutes allowed to cook to room temperature before inoculation.