MICROBIOLOGICAL EXAMINATION OF ICE CREAM SOLD IN AUCHI

BY

ADENIRAN OMOLARA MORUFAT AST/2382180770

AIBUEDEFE HAPPY AST/2382100675

A PROJECT SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL SCIENCE

LABORATORY TECHNOLOGY, SCHOOL OF APPLIED SCIENCE AND
TECHNOLOGY, AUCHI POLYTECHNIC, AUCHI, EDO STATE.

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

HIGHER NATIONAL DIPLOMA (HND) BIOLOGICAL SCIENCE LABORATORY
TECHNOLOGY, SCHOOL OF APPLIED SCIENCE AND TECHNOLOGY, AUCHI
POLYTECHNIC, AUCHI, EDO STATE.

SEPTEMBER, 2023

i
 CERTIFICATION

We certify that this project titled “Microbiological Examination of Ice Cream Sold in Auchi”

was carried out by ADENIRAN OMOLARA MORUFAT with AST/2382180770 and

AIBUEDEFE HAPPY with AST/2382100675 in the Department of Biological Science

Laboratory Technology, Auchi Polytechnic, Edo State.

……………………… ………………………
Dr. Anetehkai W.E Date
Project Supervisor

……………………… ………………………
Mr. Ohimai A. Date
Head of Department

…………………………………… ………………………
External Supervisor Date

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 DEDICATION

This project work is dedicated to God Almighty and to all lecturers in Biological Science
Laboratory Technology.

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 ACKNOWLEDGEMENT

We are grateful to Almighty God for the strength, knowledge and understanding to complete this

project. His love has been more than sufficient to keep and sustain us.

Our profound gratitude goes to our wonderful supervisor, DR. ANETEKHAI W.E for his

invaluable support, patience, time and guidance towards the completion of this research work.

Also our gratitude goes to the head of department of Biological Science Laboratory Technology

MR. OHIMAI A. for his fatherly advice. May God continue to bless and guide you. Amen

We are also grateful to our parents and family member for their entire support and love.

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 TABLE OF CONTENT
Title page i
Certification page ii
Dedication iii
Acknowledgement iv
Contents vi
Abstract viii
CHAPTER ONE
1.0 Introduction 1
1.1 Location of Study 3
1.2 Statement of Problem 3
1.3 Aim and Objective of Study 3
1.4 Scope of the Study 3
1.5 Significance of the Study 4
1.6 Limitations of Study 4
1.7 Expected Contribution To Knowledge 4
1.8 Definition of Terms 4
CHAPTER TWO
2.0 Literature Review 6
2.1 The Origin of Ice Cream 7
2.2 Nutritive Value of Ice Cream 7
2.2.1 Ice Cream Ingredient 8
2.3 Industrial Production of Ice Cream 9
2.4 Potential Microbiological Hazards 12
2.5 Health Benefit Of Ice Cream 13
2.6 Major Diseases Associated With Ice Cream 13

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CHAPTER THREE
3.0 Materials and Method 15
3.1Collection of materials 15
3.2 Sterilization of Material 15
3.3 Disinfection of Working Area 17
3.4 Culture Media Preparation 17
3.4.1 Preparation of Nutrient Agar. 17
3.4.2 Preparation of Sabouraud Dextrose Sugar 17
3.4.3 Preparation Of Inoculums And Inoculation 18
3.4,4 Purification and Storage of Isolates 18
3.5 Characterization And Identification Of Isolates 18
3.5.1 Microscopic Examination of the Isolates 18
3.5.2 Grams's Staining Techniques 19
3.6 Biochemical Tests 19
CHAPTER FOUR
4.0 Results and Discussion 22
4.1 Discussion 26
CHAPTER FIVE
5.0 Conclusion 30
5.1 Recommendations 30
REFRENCE 32

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 ABSTRACT
Ice cream is a food consumed during the hot/dry season. Its microbial quality has been critically
important to public health. This study aims at accessing the microbiological content of ice cream
sold in Auchi. Three samples were collected from three vendors (Valchi, GT fast food and Mr.
Biggs). The total bacteria counts ranged from 1.0 X 103 -3.2X103 (cfu/ml) while the fungi count
was between 1.3 X 103 -2.6 X 103 (cfu/ml). Staphylococcus spp, Bacillus cereus, Lactobacillus
spp and Streptococcus spp were the bacteria isolated while Mucor spp and Aspergillus spp, were
isolated as fungi. Some of these organisms are of public health importance because they can
cause disease to man. These microorganisms are mostly present in the materials used for ice
cream processing e.g contaminated water, container or during packaging and transportation of
the ice cream.

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 CHAPTER ONE

1.0 Introduction

Ice cream being a nutritious food serves as a good medium for microbial
infection compared to other dairy product such as cheese, sweets and yoghurt due
to the high nutritive value, almost neutral PH and long storage duration (Ojokoh,
2016).

Ice cream is a congealed diary product produced by freezing pasteurized

mixture of milk, cream, milk solid other than fat, sugars, emulsifier and stabilizers.
Product of diary origin is the main ingredients of ice cream these include whole
milk, skimmed milk, cream, frozen cream, condensed milk, product and milk solid.
Other ingredients include flavouring matters and water. Fruits nuts candles and
syrup are optionally added into ice-cream for flavor enrichment (Fotter and
Hotchkiss, 2015). Ice-cream received wide popularity and great acceptance as a
highly nutritious food internationally. (Sherikar and Majee, 2014).

Ice cream is a frozen dairy product with delicious taste and flavor and is
relished by both sex, and all age groups of easy strata of society. In all season and
in all climatic zone of the world. It is a milk product that is sold in soft and hard
form. The fat percentage in ice cream is 10% and 11% respectively, sugar may be
added up to 15% for sweetening. Glyceromonosterate is use for foaminess
(Schlundt and Jansen, 2014).

The manufacturing process and raw material used for ice cream preparation
have been previously reported as the main sources of microbial contamination and
thus deteriorate quality of ice cream (Hankin and Hanna, 2016). Primary sources of
microbial contamination into ice cream includes water and raw milk whereas the
secondary sources of microbial contamination include flavoring agents, utensils
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and the personnel associated with the processing and distribution. Sterilization
method like pasteurization followed by freezing and hardening eliminate the most
of pathogenic organisms. However a handful of hazards can still retain in the
finished products. Faulty manufacturing process, leakage in the process tanks, post
sterilization contamination, ill storage condition and low sanitary quality of
packaging materials can contribute the admittance of pathogen into the products
(Joshi and Sharma, 2004). Many psychrophiles and psychrotelerant
microorganisms like listeria manocytogenes, staphylococcus aureus, Bacillus spp,
Pseudomonas spp, Salmonella spp, Shigella spp, Streptococcus spp,
campylobacter spp, Brucella spp and coliform bacteria are known to be the
potential threats to Ice cream quality (Jay, 2012).

Commercially processed and homemade ice cream is implicated in out break

of disease. Heat stable toxin of Staphylococcus aureus caused food poisoning in
two persons through the consumption of ice cream (Akter and Ahmed, 2012). The
infection from the food handlers, contamination of the mix and contamination of
the utensil are some of the factors that are responsible for the outbreak of disease
through ice-cream. It is pertinent to mention that if any disease producing pathogen
gets entery into ice cream, it may remain viable for years in the frozen state
(Marshall, 2018).

A study on the microbial content is to indicate whether proper training on good

manufacturing practice and use of latest technologies in ice cream industries can
reduce introduction of contaminations during ice cream production and establish
the total quality management and hence guarantee public health safety.

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1.1 Location of Study

The project was carried out in Auchi Etsako west local government Area of
Edo state.

1.2 Statement of Problem

Ice cream is a nutritious food for human consumption and also an excellent
medium for the growth of many microorganism some of which may cause disease
in human beings e.g. Cholera, typhoid and bacillary dysentery contaminated ice
creams have caused several outbreaks of gastrointestinal diseases in a number of
countries in Asia, Europe and North America.

1.3 Aim and Objective of Study

 Determine and evaluate the microbiological quality of ice-cream sold in fast

food joints in Auchi.
 To identify isolated organisms from contaminated ice-cream.
 To enlighten the public and create awareness of possible side effect
associated with ice-cream.
 To determine the total aerobic bacteria and coliform counts in ice-cream.

To check the quality of the ice cream sold around Auchi, if they meet with health
standard.

1.4 Scope of the Study

The scope of the study is focused on the examination of microorganisms

associated with ice cream sold in Auchi.

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1.5 Significance of the Study

The information gathered during the process of carrying out this study will
enable individuals know the microbes that are likely to be found in ice-cream and
to examine the ice-cream sold in fast food joints around Auchi and check if it
meets up with health standard.

1.6 Limitations of Study

Some difficulties encountered during this project work include:

 Power failure.
 High cost of material was almost causing delay.
 Difficulty in getting some of the reagent.

1.7 Expected Contribution To Knowledge

Shifting consumer preference towards consumption of healthy food is

expected to support the demand of low fat ice cream in the near future. To
encourage maintenance of appropriate hygienic condition during manufacturing
and storage of ice-cream. To ensure consumer health safety to enlighten the public
and create awareness of possible side effect associated with ice-cream. To serve as
a reference material for future research.

1.8 Definition of Terms

Microorganism: is a living organism that is too small to seen with naked eye but
visible under a microscope. Also called microbe.

Psychrophiles: They are extremophilic organisms that are capable of growth and
reproduction in low temperatures ranging from -20°C + 10°C.

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Bacteria: Single-celled microorganism that can exist either as independent
organism or as parasite

Pathogen: They are small organism such as virus, or bacterium that can cause
disease.

Toxin: A poisonous substance, especially one produced by bacteria that cause

disease.

Sterilization: This refers to any process that eliminates, removes, kills or

deactivate all form of life and other biological agent such as fungi, bacteria,
viruses, spore forms, prions etc.

Pasteurization: Is a process in which package and non-package food such as milk

and fruit juice are treated with mild heat to eliminate pathogens and extend shelf.

Congealed: To change from a liquid or soft state to a thick or solid state.

Microbial Contamination: Refers to non-intent or accidental introduction such as

bacteria, yeast, mould, fungi or their toxins and by products

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 CHAPTER TWO

2.0 Literature Review

Ice-cream a milk base product, is an important commodity in the

international trade as it is consumed globally in Bangladesh where the summer is
the longest season, varieties of ice cream serve as one of the excellent and popular
ready-to eat food product among children and adults as well. Ice cream is known to
be frozen diary product usually consisting of milk, cream milk, sugars, egg,
emulsifiers and stabilizers, flavouring agents, fruits, nuts, candles syrup and other
additives. Proper pasteurization of ice cream mix and hygienic handling and
processing of mix following pasteurization are of pivotal significance to have a
good quality of ice cream (Willey et al., 2017). In order to ensure safety, it is
imperative that the public and trade are advised to pay adequate attention on some
key areas from the preparation to the consumption of ice cream. Moreover,
emphasis is given on the introduction of Haccp. In milk processing establishment
from food safety point of view (Anderson and Nielson, 2015). Even with an
apparent nutritive image, ice-cream have been often reported as the leading vehicle
of microbial infection compared to the other diary products. The presence of
coliform in ice cream may be due to post pasteurization contamination by handler
with poor sanitary practices. The level of the presence of this organism in food has
been described as index of food hygiene. (Sarker et al., 2013).

Indians are very fond of milk and milk product, a wide variety of milk products
such as butter, cream dried milk, butter milk and ice cream etc. are manufactured
and sold in the markets (Sherikar and Majee, 2014). Moreover, india is the largest
producer and consumer of milk and milk product in the world.

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2.1 The Origin of Ice Cream

During the 5th Century BC, Ancient Greeks ate snow mixed with honey and
fruit in the market of Athens. The father of the modern medicine, Hipocrates
encourage his Ancient Greek patients to eat ice " as it livens the life. Juices and
increase the well being" (Graham and Moturin, 2014).

In 400 BC, the Persians invented a special chilled food, made of rose water
and vermicelli, which was serve by royalty during summers. The ice cream was
mixed with saffon, fruits and various other flavor (Rhaman, 2017). It frozen
mixture of milk and rice was used in China around 200 BC. They poured a mixture
of snow and saltpeter over the exterior or container filled with syrup for in the
same way as salt raises the boiling point of water it lower the freezing point below
zero (Warke et al., 2019).

In the sixteenth century in India, the Mughal emperors used relay of

horsemen to bring ice from the Hindi Kush to Delhi were it was used in fruit
sorbets. Kulfi is a popular frozen diary dissert from the Indian subcontinent and is
often described as traditional Indian Ice cream. It originated in the sixteenth
century in the Mughal (Marion, 2018).

2.2 Nutritive Value of Ice Cream

The composition of Ice cream is usually expressed as a percentage at it

constituents, for example, the percentage of milk fat, milk solid not fat, sugar, egg
solids, stabilizers which are the compound added in very small quantities to
strongly influence the formation and growth of ice crystal so as to render the
product with desired body and texture. (Hossain and Kabir, 2012).

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The milk protein contain in ice cream are of excellent biological value, because so
they contain all the essential amino acid. Protein in the diet supply the amino acid
required for the growth of infants and children and for maintenance of tissue in
adult. Lactose constituents over one-third of the solid matter in milk and 20 % of
the carbohydrate in ice cream which makes the products suitable for lactose-
intolerants people (Bigalke and Chappel, 2012).

Fat is of major importance in ice cream. It contribute a rich flavor and is a good
carrier for added flavor compounds and promotes desirable texture qualities.

Milk fat contributes significantly to the nutritive value of ice cream. It is a rich
source of energy, serves as a source of fat-soluble vitamin A, D, E and K and
contains significant amount of fatty acid. Milk and milk product like ice cream are
the richest sources of calcium, phosphorous and other mineral are essential in
adequate-nutrition. Ice cream is rich source of many of the essential vitamin
without which normal growth and health cannot be maintained (Yaman et al.,
2016).

2.2.1 Ice Cream Ingredient

Ice cream is composed of a mixture food ingredient like milk product,

sweetening materials, stabilizers, color, flavours, egg products and emulsifiers.

Emulsifiers: Emulsifiers are used to produce ice cream with a smoother body and
texture, to impact dryness and to improve whipping ability of mix emulsifiers
extensively use are monoglycerides or diglycerides, or sorbates and polysorbates
and are added at the rate of 0.1-0.4% of the fininsh product, egg yolk solid are also
used as emulsifiers.

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Flavours: Flavours is generally considered as one of the most important
characteristics of ice cream the kind of flavouring material influence the quality of
the ice cream mix since slight of-flavor in it can obscure the delicate flavor of the
flavouring materials to be added (Willey et al., 2013).

Colours: Ice cream should have a delicate attractive color that can be readily
associated with the flavors. Most colors are of chemical origin. Colors are
available in liquid or powder form (Oranusi et al., 2013).

Sugar: Sugar gives ice cream a sweet taste, but that is not its most important role.
The sugar controls the amount of ice formed during the freezing of the product.
This ensures that the ice cream is soft enough to scoop and eat. It also gives
thickness to the product and makes it stable to heat shock (being in and out of the
freezer). The sugar will help as well to release the flavor of the ice cream. An
average ice cream contains 16-23 grams sugar per 100g. This means around 11-16
grams in typical serving. The sugar contributes up to 50% of the energy in the
product.

Milk Protein: Milk protein are a key ingredient of ice cream products. The protein
in ice cream ensures small fat and air bubbles are produced. They give ice cream
it's creaminess. The protein also contributes to the flavours. Protein in ice cream
deliver about 2-12 % of energy (average 8%) in the product.

2.3 Industrial Production of Ice Cream

The production of ice cream include many steps classified under the
following main parts.

 Ice cream mix making (making of ingredients pasteurization and

homogenization)

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  Soft ice-cream production (aging and freezing).
 Hard Ice-cream production (packaging and storage) the manufacturing of ice
cream mix involves mixing of ingredients, pasteurization and
homogenization. For soft ice cream, premade ice-cream, premade ice-cream
mix is supplied to the retail outlets under refrigerator (7 OC) aging and
freezing at around -5° C are performed in the vending machines at theretail
level. Other ingredient such as fruits and nuts, may be added to the soft ice
cream at the time scale for flavor enrichment in frozen confection factories
the soft ice cream will be packed and harden too produce the hard ice cream.

The following described the technological details of each processing step

(Cheesbrough, 2006).

Mixing of Ingredient

The first step of preparing ice cream mix is to combine the liquid ingredients
and heat then to around 43° C, then sugar and other dry ingredient, except nuts and
fruits are added to the lukewarm mix. (Marshall, 2018)

Pasteurization

The mixture is then pasteurized by a healthy process, either in batch or

continuous modes depending on the production size. Pasteurization temperatures
for ice cream mix around 70OC for 10-30 minutes are higher than for a plain milk
because high fat and sugar content, tend to protect bacteria from heat treatment
(Bell and Kyriakides, 2018).

Homogenization

The pasteurized mix is then homogenized. High presence of 4.1 X 106 Pa

(600 Psi) to 1.7 X 107 Pa (2500 Psi) are use to break down fat globules.
10
 This can clump fat globules and together with added emulsifiers can prevent
churning of fat into butter granules (milk fat) during freezing step.
Homogenization also improves the texture of ice cream. After that the
homogenized mix is cooled down to 40OC for further processing. For production of
soft ice-cream using vending machines (Walstra, 2019).

Aging

Aging is the first step in soft ice cream production. The mix is held in
sterilized vats from 3-24 hours at temperature of around 4 OC or lower. It allows
some physical changes such as fat crystallization adsorption of protein onto fat
globules and uncensement of the mix's viscosity. These changes lead to a quicker
whipping to the desired overrun in freezing process, smoother ice-cream body and
texture and slower ice cream meltdown (potter and Hotchkkiss, 2015)

Freezing

During air is incorporated in the mix and cool down to around 5 OC. Freezing
must be performed as quickly as possible to prevent the information of large ice
crystals. The air cells in ice cream should be small and evenly distributed in order
to maintain stable frozen foam. At this low temperature -5 OC not all water particles
are crystallized and therefore, ice cream is only in semi solid state. The semi-sold
ice cream emerging from the freezer is similar to the constituency of soft ice cream
produced at the retail fevel (Kambamanoli-Dimov, 2018).

Packaging

To produce ice cream, the semisolid ice cream is packed into cartons drums
for hardening to form specific shape of ice cream products and to have longer shelf
life (more than one year) (Capplicino and Sheroman, 2016).

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Hardening

The pre-packed semisolid ice-cream is then placed in a hardening room

where the temperature of about -34OC is maintained. The low temperature keeps
the core temperature of ice cream at around -18OC.

Hardening should be performed quickly, to prevent the formation of large

ice crystals and to maintain better quality of ice cream (Ikenebomeh and Ogaguvia,
1993).

Storage

After hardening, the hard ice cream will then be placed in the cold store
rooms with the temperature of around -18 OC. from this storage, the hard ice cream
must be kept at -18'C or below throughout storage, transportation and display
(Azadina et al., 2011).

2.4 Potential Microbiological Hazards

Ice-cream, a milk based product is a good media for microbial growth due to
high nutrient value, almost neutral pH value (PH -6-7) and long storage duration of
ice cream (Bell and Kyriakides, 1998). However pasteurization, freezing, and
hardening steps in the production can eliminate most of the microbiological
hazardsJce cream must be heat treated during the production process. Ice cream
may also serve as good medium for the growth of many organisms. Some may be
Pathogenic to human and others can cause the spoilage of the product, the potential
microbiological hazard found in ice cream can be introduce after the pasteurization
through the addition of contaminated ingredient and improper handling (Vougnt
and Tatini, 2012).

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2.5 Health Benefit of Ice Cream

- Source of vitamins.

lce cream is a good source of vitamins A. B-6, B-12, C, D and E. It apparently

contains vitamin K, which prevents blood clothing.

- Provides Energy

Not only that ice cream have nutritive value, it also is an incredible source of
energym

- Source Of Minerals

Minerals like calcium and phosphorus are found in ice cream. Calcium is an
essential mineral for us to maintain strong bones and reduces the chance of kidney
stones. Mood swings and PMS can also be prevented by eating ice cream.

- Stimulates The Brain

lce cream stimulates the thrombotonin, which is a hormone of happiness and helps
reducing the levels of stress in the body. Ice cream is made of milk which contains
L-triptophane, which is a natural tranquilizer and helps in relaxing the nervous
system. It also helps to prevent symptoms of insomnia.

2.6 Major Diseases Associated With Ice Cream

There are a number of microbes that are of concern to the ice cream and
these are at the heart of food safety regulations imposed not only manufacturer
nationally but by vendors as well. Whilst some microorganisms can affect ice
cream quality, the most significant concern are organisms as indicators of hygiene
and potential human pathogens.

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 - E.coli and other coliforms.
- Listeria spp.
- Salmonella spp.
- Staphylococcus spp.
- Pseudomonas spp.

Commercially processed and homemade ice cream is implicated in outbreak

of disease. Salmonella enteritidis contamination of ice cream was associated with a
1996 multistate outbreak. Heat stable toxins of Staphylococcus aureus caused food
poisoning_in two persons through the consumption of ice cream (Akter and ,
Ahmed, 2012).

The type of milk and milk products (raw milk and condensed milk etc)
Sanitary level of equipment efficiency of pasteurization and health of worker may
Contribute to the microbiological aspect of ice cream. Sugar is contaminated with
coliform, thermophilic, mesophilic, fungi (mould and yeast) which gain entry
during production and storage may result in disease outbreak. Packaging are
reported to be source of fungal spores and hence, it is advised to effectively
sanitize the packaging materials before using to pack ice cream (Ojokoh, 2016).

The same danger of illness caused by drinking raw milk are inherent in ice
cream either made irom raw milk and cream or handled under unsanitary
conditions. Ice cream has been incriminated as a transmitter of pathogenic bacteria,
but outbreak of disease due to commercially manufacture, ice cream are rare. At
various times, ice cream has been found to habour typhoid fever, paratyphoid
fever, diphtheria and Scarlet fever organism (Prescott et al., 2018).

Many major food poisoning outbreaks have cause by human contamination.

One in 1945 was due to Staphylococci carried by a worker in the cook house of an
14
army hospital, which were introduced into batches of ice cream mix after the
ingredient has been cooked. Moreover around 700 people were affected by a
staphylococcal toxin which develop during that period. (Akter and Ahmed, 2012).

2.7 Steps for Preventing Microbial Infection In Ice Cream

1. Beelean and tidy.

2. Clean protective clothing e.g. apron or overall should be worn where
appropriate, particularly advisable in business serving large quantity of ice
cream e.g. Ice cream parlours.
3. Keep hair clean and neat and wear hats or hair nets which effectively contain
the hair.
4. Keep finger nails short and clean.
5. Cover cuts, sores, or grazes with coloured waterproof dressing.
6. Do not smoke, consume food, chew gum, lick fingers, cough or sneeze
where ice cream is being prepared or served.
7. Do not serve ice cream if you are suffering from diarrhea, vomiting,
jaundice, sore throat with fever, infected skin lesions/cuts on exposed body
parts, discharges from eyes, ear, nose, mouth/gun.
8. Always sanitize packaging materials before using to pack ice cream.
9. Hands are common means of transferring bacteria to ice-cream, therefore it
should be washed and kept clean at all times.

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 CHAPTER THREE

3.0 MATERIALS AND METHOD

3.1 Collection of materials

Samples of ice cream were purchased from three different sales outlet in
Auchi which include Valchi fast food labeled as (A), Mat Ice (B) and GT Plaza
(C). Samples were aseptically collected in a clean polyethelene bag and transferred
immediately to the microbiology laboratory of Auchi Polytechnic Auchi for
microbiological analyses as described by the methods of Fawole and Oso (211).

Materials

The ice cream samples were collected from three sales outlets in Auchi,
other materials used are Nutrient agar, Sabouraud dextrose agar, measuring
cylinder, petri dishes, Beaker, 10ml Pipette, flat bottom flask, weighing balance,
Test tubes, test tube racks, Autoclave, Incubator, Slide, Microscope, cotton wool,
Distilled water, Spatula, Wire loop, crystal violet, Lugos iodine, Acetone alcohol,
Safranine. Phenol red, Kova's reagent, Glucose sugar, Sucrose sugar, Lactose
sugar, Maltose sugar, Durhan tube, Spirit lamp, Ethanol and Lactophenol in cotton
blue stain.

3.2 Sterilization of Material

All glass wares were first washed with detergent and rinsed with distilled
water, wrapped with aluminum foil after drying and sterilized by dry heat method
in the oven at a temperature of 160OC for 2-3 hrs.

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3.3 Disinfection of Working Area

The working area were disinfected thoroughly before and after use with
ethanol (75%V) cotton wool was soaked in ethanol and used to clean the working
bench, a Bunsen burner was put on and the flame was allowed to burn , this helped
in sterilizing the air in the laboratory.

3.4 Culture Media Preparation

The media used in this study is nutrient Agar and Saboraud Dextrose Agar.
The media was prepared according to manufacturer's specification.

3.4.1 Preparation of Nutrient Agar.

28g of nutrient agar powder was weighed using a weighing balance and
dispensed into a beaker, 100mls of distilled water was measured using a measuring
cylinder and dispensed into the beaker containing the agar powder, it was stired to
dissolve for 10mins. The mixture was transferred into a conical flask and the neck
of the flask was corked with cotton wool wrapped in aluminum foil. It was
autoclayed at a temnperature of 121 OC and pressure of 1SPsi for 15-20minutes.
The sterilized agar was allowed to cool to about 45°C and then aseptically poured
into perti dishes and allowed to set. (Cheesbrough, 2016).

3.4.2 Preparation of Sabouraud Dextrose Sugar

65g of sabouraud dextrose agar powder was weighed using weighing

balance and dispensed into a beaker. 1000m/s of distilled water was measured
using a measuring cylinder and dispensed into the beaker containing the agar
powder, it was stirred to dissolve for 10mins. The mixture was transferred into a
conical flask and the neck of the flask was corked with cotton wool wrapped in

17
aluminum foil. It was autoclave of 120 OC and pressure of 15Psi for 15-20minutes.
The sterilized agar was allowed to set (Cheesbrough, 2006).

3.4.3 Preparation of Inoculums And Inoculation

10g of each ice cream sample was weighed and mixed with 9mls of water to
get the mother sample; serial dilution was carried out on the samples after which
the innocula were aseptically transferred into plates of the nutrient media using the
streak plate techniques. A sterile wire loop was used to take an aliquot of the
inoculum and aseptically transferred to the plates and streaked.

3.4,4 Purification and Storage of Isolates

Bacterial colonies that appeared on the primary isolation plate were sub-
cultured onto fresh nutrient agar plates to obtain pure cultures of the different
isolates. The final cultures containing discrete colonies were transferred onto slant
made with McCartney bottles and test tubes containing nutrient ayar. The slarits
were stored in the refrigerator at 40OC for further studies.

3.5 Characterization And Identification Of Isolates

The bacteria isolate from different samples were grouped on the basis of
colonial morphology. The criteria used, were the size of the colony, color, surface,
edge andelevation as described by (Cheesbrough, 2016).

3.5.1 Microscopic Examination of the Isolates

Smears of each of the different isolates were made with a clean grease free
slide and air dried. The bacterial isolates were stained using Gram's Method of
staining to study the morphological appearance of the cell as well as the Gram
reactions (whether they are Gram positive or Gram negative) and cell arrangement.

18
Gram positive cells retained the purple color of the initial dye while the Gram
negative cells retained pinkish-red color of the counter stains (Ikenebomeh and
Ogagovia, 1993).

3.5.2 Grams's Staining Techniques

A thín smear each of the pure 24 hours old culture were prepared on clean
grease-free slides, allowed to air-dry then fixed by passing swiftly over a burning
flame. The slides containing the smear were tlooded with crystal violet solution for
60sec, rinsed with water and blot to remove water residue. The smear again were
flooded with lugos iodine which acts as a mordant for 60secs, rinsed with water
and bottled. They were then decolorized with acetone alcohol for a few seconds
and rinsed after which they were counter stained with safranine for 30-60secs
washed.With water and kept to air dry before they were observed under the oil
immersion objective of the microscope. Gram positive cells appeared purple whle
Gram negative appear pink or red.

Catalse Test

The enzymes catalase mediates the breakdown of hydrogen peroxide into

Oxygen and water. The presence of the enzyme in a bacterial isolates is evident
when a small inoculum is introduced into hydrogen peroxide, and the rapid
elaboration of oxygen bubbles occurs. A loopful of 24hours old culture was placed
on a clean slide and a drop of 3% hydrogen peroxide was placed on it. Gas seen as
white bubbles indicates the presence of catalase enzyme which is a positive result.

3.6 Biochemical Tests

Biochemical tests were performed to confirm the identity of the isolated

bacteria 24hours old cultures were used to perform the biochemical test.

19
Coagulase Test

Coagulase is an enzyme-like protein and causes plasma to clot by converting

fibrinogen to fibrin, Coagulase test is used to differentiate staphylococcus aureus
(positive) which produce the enzyme coagulase, from s. epidermis and s.
Saprophiyttcus (negative) which do not produce coagulase.

A drop of physiological saline was placed on two separate slides, using a

wire loop a portion of the isolated colonv was emulsified in each drop to make two
thick suspensions. A drop of human plasma was added to one of the suspensions,
and mix gently. Look for clumping of the organism within 10seconds. No plasma
is added to the second suspension to differentiate any granular appearance of the
organisms from true coagulase clumping

Indole Test

This test demonstrates the ability of certain bacteria to decompose the amino
acid tryptophan to indole isolated cultures were inoculated into peptone broth
medium incubated at 37'C for 48 hours after which 5 drop of Kovac's reagent was
added to the culture. The appearance of a red ring indicates indole production.

Sugar Fermentation

The carbohydrate fermentation test is used to determine whether or not

bacteria can utilize a certain carbohydrate. It test for the presence of acid/or gas
produced from the fermentation of a single particular carbohydrate.

- 0,05% to 1% of desired carbohydrate was added to all flask containing

peptone water.
- Durhamn tubes were inverted in all tubes, the Durham tubes were fully filled
with the broth.
20
 - It was sterilized at 115'C for 15 minutes.

Note: Do not overheat the phenol red carbohy drate fermentation broth the
overheating will result in breaking down of the molecules and form compounds
with a characteristics color and flavor. The process is known as caramelization of
sugar. (The browning of sugar).

- The tubes were labeled properly.

- Each labeled carbohydrate broth was aseptically inoculated with bacterial
culture (keep uninoculated tubes as control tubes).
- The tubes were incubated at 18-24 hours at 37OC.
- The reaction was observed after 24 hours.

A color change from red to yellow indicate a positive test, while a retention of
the red color is negative. When the Durham tubes move up it indicates gas
production.

Identification of Fungi Isolates

The sabouraud dextrose agar plates were cultured at room temperature for 4-
5 days after which the colonial characteristics were observed and lactophenol in
cotton blue stain was used to stain the growth and observed under the 10 X
objective.

21
 CHAPTER FOUR

4.0 Results and Discussion

The result of the microbiological examination of ice cream sold in Auchi are
presented below:

Table l: total bacterial count (cfu/ml) isolated from ice cream samples.

Samples Dilution No. of colonies on Total number of colony

factor plate forming unit (cfml)

A 103 13 1.3 X 103

B 103 32 1.3 X 103

C 103 10 1.3 X 103

22
 Table 2: Result of Cultural, morphological & biochemical test of bacteria isolate from different ice-cream sample.

Isolate Cultural Morphological Gram Catalase Coagulase Indole Sugar Fermentation Isolated
Characteristics Arrangement of cells Reaction Test Test Test Organisms
Glu Lac Mal Suc
A (Zaris Transperent, Smooth Cocci arranged in + - - - + + + + Streptococcus spp
Fast and round colonies chains
Food)
Moist irregular Bacillis arrangement + - - - + + + + Lactobacillus spp
growth in paris and chains
Gray, granular Bacillis with central + + - - + - + + Bacillus cereus
spreading colonies spores after 48hrs or
with a rough malted culturing
surface and irregular
perimeters

B (MAT Yellow to white Cocci arranged in + + - - + + + + Staphylococcus

ICE) color, mucor-liked clusters spp
on nutrient aga
Transperent, Cocci arrangement + + + + + Staphylococcus
smooth and round in chain pairs and spp
colonies in chain
Moist irregular Bacillis arranged + - - - + + + + Lactobacillus

23
 growth in pairs and in spp
chain
C Gray, Granular Bacillis with + + - - + - + + Bacillus cereus
spreading central spores
colonies with a after 48hrs of
rough malted culturing.
surfacesa rough
malted surfaces
and irregular
parameters
Moist irregular Bacillis arranged + - - - + + + + Lactobacillus
growth in pairs and chains spp

24
Table 3: Microorganism isolated from ice cream samples

Sample Isolated Organisms

Sample A Bacillus cereus, Lactobacillus spp and Staphylococcus spp
Sample B Staphylococcus spp, Lactobacillus spp and Staphylococcus
spp
Sample C Bacillus cereus and Lactobacillus spp

Table 4: Plate count for fungi isolated from ice cream samples.

Samples Dilution Plate count (sfu/ml)

factor
A 103 1.3 X 103

B 103 1.3 X 103

C 103 1.3 X 103

Table 5: Cultural and morphological characteristics of fungi isolated from ice

cream samples

S/N Cultural Characteristics Morphological Suspected

Characteristics Organisms

1 Whitish filamentious Absence of rhizoids Mucor spp

mycelium PDA branched sporangiosphore
present

2 A yellow green colony Rough wall condiospore, Aspergillus spp

rough texture on PDA short chained candida,
septate hyphae

25
Table 6: -Occurrence of fungi isolated from ice cream samples

Samples Aspergillus spp Mucor spp

A + +

B + +

C + +

4.1 Discussion

Table 1 represents the average plate count for the samples analyzed, sample
A had a plate count of 1.3 X 10 while sample B and C had plate count of 3.2 X 10³
and 1.0 X 10' respectively with the highest count of 3.2 X 10³ for sample B and the
lowest was 1.0 X 10 obtained for sample C. The result obtained in this study
represents the current status of the microbiological quantity of ice cream being sold
in fast food within Auchi.

Different species of bacteria were identification from the ice cream under
study as revealed in table 2 and some of these organisms are of public health
importance. This could be because these microorganisms are mostly present in the
materials used for ice cream processing e.g. water used, may be from contaminate
driver and some of the microorganisms commonly present in the soil could get to
the ice cream by contamination.

Streptococcus spp. was isolated from all samples except sample C. Though
streptococcus spp. acts as probiotic, the only probiotic in the streptococcus group
is streptococcus thermophillus (Oranusi and Olarewaju, 2013). The isolation
indicates favourable environment within the ice cream capable of promoting the
growth of these organisms. The presence of these organisms is of health

26
significance as some of them may be capable of causing various illments of man
which can be fatal (Okojoh, 2016).

Staphylococcus aureus was isolated from sample B. The absence of s.aureus

has been rarely reported in several studies (Marshall, 2018). The presence of
s.aureus may have resulted from either insufficient pasteurization of milk, or
human exposure (Yaman et al., 2016). In humans, the main reservoir of s.aureus is
the nasal cavity. The organism finds their way to the skin and into wounds directly
or indirectly. The most common skin sources are arms, hands and face. In addition
to skin and nasal cavities s.aureus may be found in eyes, throat and intestinal tract
(Yaman et al., 2006) from these sources, the organisms finds its way into air and
dust, onto clothing and in other places, from where it may contaminant food (Jay,
1996). There are other sources like coughing, talking and sneezing which produce
droplets. These droplets could settle on ice cream during transportation, storage
and retailing (Ojokoh, 2016). S.aureus which is mainly transmitted from man and
animals can lead to staphylococcal food poisoning as a result growth of the
organism and release of enterotoxin in the food.

Bacillus cereus was obtained from two samples which are sample A and C.
The presence of these pathogenic organisms in the analyzed ice-cream should be
viewed with concern by the consumers, producing company and the government
since food poisoning by Bacillus spp. is possible through consumption of
contaminated ice cream. The proper handling of ice cream is very important both
during manufacturing and storage to prevent outbreak of food borne diseases
sources of contamination could be as a result of contamination from various
sources notable handlers during preparation especially under unhygienic condition
also from retailers, most of them do not store ice cream at the appropriate storage
temperature. Even the refrigerated samples are not always maintained between 40°

27
C and 60OC due to unstable powers outage. (Prescoot et al., 2019) reported that
refrigeration at 50OC retards bacterial growth. High temperature therefore favours
and encourages bacterial growth which eventually causes spoilage.

Bacillus species and other thermoduric bacteria are spore formers and can
become part of some of the ingredients of the ice cream mix, and thus can persist
after pasteurization. Bacillus species are common environmental contaminants and
has been shown to be transient micro-flora of the hand and surfaces due to its spore
forming ability (Oranusi et al., 2013). Bacillus species have been implicated in
humarf pathogenesis and as food spoilage organisms.

Lactobacillus is indispensable to the food and diary industry. They are

usually not pathogenic (Willey et al., 2018). Lactobacilli, a member of the lactic
acid bacteria, constitute a member of the probiotic bacteria. It has been established
that according to the environmental illness resources, the benefits that lactobacillus
can provide include; preventing and treating diarrhea caused by antibiotics;
preventing of virginal and urinary tract infections; preventing of overgrowth of
bacteria like Helicobacter pyroli, salmonella and E.coli, and helping with the
digestion of the normal flora of the human body in the mouth, intestinal tract and
vagina. They are sometimes responsible for the spoilage of beer, milk and meat
because their metabolic products contribute undesirable flavours and odors (Willey
et al., 2018)

Aspergillus spp and mucor species were isolated from all samples and study
it should be noted that some species of Aspergillus are known to produce
mycotoxins which are harmful to man, thus their presence in ice cream is
undersirable (Ojokoh, 2006). They have been implicated in superficial cutaneous
sub-cutaneous and systematic mycoses (Oranusi et al., 2013). Their presence in all

28
samples may pose a great risk for public health. The presence of these organisms
indicates contamination from handlers during processing and the micro-flora load
could be attributed to the rate of exposure during sale (Jay, 2016).

29
 CHAPTER FIVE
CONCLUSION AND RECOMMENDATION
5.0 Conclusion
In conclusion, although milk is sterile as it leaves the udder of a healthy
cow, it can be contaminated during handling in the farm by a variety of organisms.
Ice cream being a favorite food to many Nigerians and a delicacy rich in glucose,
protein and fat, can be a source of infection if not properly pasteurized and
handled.

Both the total bacterial plate count and the total fungal count in all the
categories of ice cream were low. However the isolation of Bacillus spp.
Streptococcus spp, Lactobaciluus spp, and Staphylococcus spp suggest that ice
cream could be a source of infection to humans particularly children and
vulnerable elderly people if the counts are higher than normal.

5.1 Recommendations

Meat pie should be prepared hygienically to eliminate microorganisms that

might contaminate the. The milk handlers (vendors) should be given a proper
orientation on how to produce ice cream under aseptic techniques, thus the milk
mixing, pasteurization, homogenization, aging and storage. The utensils such as
milk pail, bulk containers should be properly washed with clean water. The
vendors should ensure that the ice cream is not exposed to air without cover for
flies and blowing air will deposit air borne pathogens into the ice cream.
Consumers should always insist on purchasing properly stored products since the
problem of the bacteriological quality lies mainly in handling and storage
conditions . The government should make it a policy that all animals used for diary
production especially the cows be vaccinated against Mycobacterium spp every

30
year since this specie of organism are responsible for Mycobacterium bovis of
cattle and Mycobacterium tuberculosis in humans.

Consumers are encouraged to buy freshly prepared ice cream and not
packaged / transported ice cream that may thaw due to change in temperature
which may encourage growth of microorganisms.

31
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