ISOLATION AND CHARACTERIZATION OF BACTERIA

FROM DIFFERENT FOODS

By

RAQIB SHAH SYEDZADA

ROLL NO 30689

Bachelor of Science in biochemistry

DEPARTMENT OF BIOCHEMISTRY
FACULTY OF HEALTH SCIENCES
Hazara University MANSEHRA
Session 2013-2017

1
2
 ISOLATION AND CHARACTERIZATION OF BACTERIA
FROM DIFFERENT FOODS

A thesis submitted in partial fulfillment of the requirement for the Degree of

Bachelors of Science in Biochemistry

Submitted B y RAQIB SHAH SYEDZADA

Supervisor Mrs.NODIA SHUJAAT

Assitant prof.
Department of biochemtry
Hazara University, Mansehra

Bachelor of Science in biochemistry

DEPARTMENT OF BIOCHEMISTRY
FACULTY OF HEALTH SCIENCES
HAZARA
University MANSEHRA
Session 2013-2017
3
 APPROVAL CERTIFICATE

This thesis is entitled isolation if bacteria from different food products

submitted by Raqib shah syedzada is accepeted in its present form by the department of
biochemistry,Faculty of Health sciences, Hazara university ,Mansehra,as fulfilling the thesis
requirement for the degree of Bachelors of sciences in Biochemistry

Approvel Comittee

1.Mrs.Nodia Shujaat
Supervisor ____________________
Assistant prof.
Deparment of Biochemistry

2.Dr.Mukhtiar Hassan
Dean Faculty of Health Sciences _____________________
Chairman Department of Biochemistry

DEPARTMENT OF BIOCHEMISTRY
FACULTY OF HEALTH SCIENCES
HAZARA
University MANSEHRA
Session 2013-2017
4
 ISOLATION AND CHARACTERIZATION OF BACTERIA
FROM DIFFERENT FOODS

DECLARATION

The work presented in this guess was carried out by me under the supervision of Madam Nodia
shujaat. The conclusions are my own research after numerous discussion with my supervisor and
the thesis is my own composition. I have not presented any part of this work for any other
degree. I testify to the best of my knowledge that the above declarations are true.

RAQIB SHAH SYEDZADA

DEPARTMENT OF BIOCHEMISTRY
FACULTY OF HEALTH SCIENCES
HAZARA
University MANSEHRA
Session 2013-2017
5
 THESE ARE THE COMMUNICATIONS OF
ALLAH WHICH WE RECITE TO YOU WITH
TRUTH AND ALLAH DOES NOT DESIRE ANY
INJUSTICE TO CREATURES
(AL Quran)

6
 This humble effort is dedicated to my dearest
Parents, Brothers, Sister, Teachers, my best
friends & one of my special TEACHER Mrs.
Nodia shujaat Who made me what I am at
present and encouraged me in ups & downs of
life & keep me motivated to strive hard in every
walk of life.

7
 TABLE OF CONTENTS

Page N0

ACKNOWLEDGMENT 10

LIST OF TABLES 12

LIST OF FIGURE 11

LIST OF ABBREVATIONS 13

ABSTRACT 14

CHAPTER 1 15

Introduction 15
1.1 Introduction of microorganism 15
1.2 Types of microorgaism 16
1.3 Importance of microorganism 17
1.4 Function of microorganism 18
1.5 food microbiology 19

1.6.: status of food microbiology

1.7: development 20
1.8: title of study 21
CHAPTER 2 22
Review of Literature 22
CHAPTER 3 26
Material and methods 26
3.1: Equipmentt
3.2: Material
3.3: sample collection 27
3.4: Bacteriological Analysis
3.5: Morphological Characterization
8
 3.6: Biochemical Characterization
3.7: Media preperation 28

3.7.1:Sample preperation
3.7.2:Total vible count 29
3.7.3: subculturing 29
3.7.4: identification
3.8: Gram Staining 30
3.8.1: catalase 30
3.8.2: Methyl Red 30
3.8.3: Citrate Utilization 30
3.8.4: Oxidase Test 32
3.8.5: indole Test 32
CHAPTER 4 33
Results and Discussion 33

Morphological Analysis 46
Summary 45
6. References 48

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 ACKNOWLEDGEMENT

I am highly indebted to Almighty Allah, The compassionate and The Merciful, who has
enabled me to complete this report.

I would like to pay my deep regards to all my teachers of biochemistry

I would like to pay to Special thanks to Ms.Nodia Shujaat (Assistant professor) who
provided me facilities to conduct Research in islamabad. for giving me confidence to conduct
Research work and for her precious guidance

Special appreciations to Ms.Nodia Sohoujat (Assistant professor), Dr. Sajid Tanoli,

Dr. Nighat sultana Ms. Saima ,Sir adeel, sir imran ullah,Madam Attiya from HEALTH
SCIENCE Biochemistry. They were also extremely kind & affectionate to me. Their guidance
and valuable discussions are gratefully acknowledged.

I am intensely thankful to my "PARENTS" for their encouragement, help and

cooperation. My Parents motivated me at the times of load and relieved me from tension.
Without their continuous love and support, I would not have been able to complete my
placement.

Thanks to my very nice Brother like friends Mr. Abdullah, Saddam ullah ,Saad
gowati,Sijjad urehman ,Hammad and Asif,atif naveed,kalim.Anees

I am truly grateful to Hena,Saba,asma,Zarka and my cousins specially junaid Hussain

and Mumtaz Hussain

Once again I am presenting my deep gratitude toward my Abba & Amma and to the one
who always been a constant source of inspiration to me syed Bakhtiar shah .I Am Deeply
indebated to my all family members Abdul Ghafar shah,abdul basir shah,

RAQIB SHAH SYEDZADA

10
 LIST OF FIGURES
Figure.1 Microbe Help full and Harmfull

Figure.2 Gram bacteria in food samples on basis of staining

Figure.3 prevalance of Basillus,klebsiella,pseudomonas,E.coli,lactobacillus

Figure.4 prevalance of bacteria in fruits

Figure.5 prevalance of bacteria in vegetables

Figure.6 prevalance of bacteria in Meat

Figure.7 prevalance of bacteria in Milk

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 LIST OF TABLES

Table 01 Sample type and code

Table 02 Morphological Characterization of Bacteria

Table 03 Biochemical Characterization of Isolate

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 LIST OF ABBREVATIONS

1 NARC National Agricultural Research Centre

2 FSPDI Food Sciences Product Development Institute

3 IMVIC Idole test,Methylated Red Test,Voges

Proskan,Citrate Utilization.

4 Ssp. specie

5 CLED Cysteine,Lactose-electrolyte-deficient

6 TVC Total Vibal Count

7 PDA Potato Dextrose Agar

8 T.B Tuber closis

9 E.Coli Escherichia coli

10 N.R Nitrate Reduction

11 CFU Colony forming unit

12 S Sample

13 I isolate

14 g gram

15 rpm round per minute

16 ml millilitre

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Abstract

Bacteria are the main and an important cause of food spoilage. They present every
where food and water are present and the temperature is suitable. To resist harm, some bacteria
can form spores tough reproductive cells that are able to survive under adverse conditions, that
can resist damage by heat as in cooking, by cold as in freezing and by chemicals such as
disinfectants. Bacteria need four hours to adapt to the new environment. 04 food samples were
collected from CHAKSHEZAD islamabad and 3 bacterial isolates viz. , Klebsiella,
Pseudomonas, E.coli, were isolated and characterized on the basis of morphology and
biochemical reactions. It was found that the , Klebsiella and Pseudomonas were the dominating
species in the spoilage of every categories of food material. In fruits, vegetables and meat
Klebsiella was the most potent spoiling bacteria. In meat, milk and in milk products
Pseudomonas was found in most of the spoilages. Although Klebsiella, Pseudomonas and
Bacillus were dominant in spoilages. The bacteria especially gram negative, was key responsible
for food spoilage

14
CHAPTER- 01

INTRODUCTION

Microorganisms or microbes are living organisms whose size is so small that they are usually
not visible to naked human eye. Microbes have adapted to inhabit almost every corner of the
world. They live in the oceans and lakes where they provide a valuable food source for larger
organisms. They live on land where they may cause the decay of dead organic material and
recycling of valuable nutrients. Many even live on or within other larger living organism
where they may help or hinder their bodily functions.

Microorganisms or microbes are microscopic organisms that may exist as unicellular,

multicellular or in the form of cell clusters. They can be divided into six major types: bacteria,
archaea, fungi, protozoa, algae, and viruses.

Humans have several reasons to be interested in the study of microorganisms. Many

microorganisms cause disease in humans. Bacteria and fungi can be parasites of humans, causing
anything from food poisoning to athletes foot to malaria. All viruses are pathogenic, or disease-
causing. Viruses are responsible for deadly diseases such as AIDS and polio as well as milder
forms like the common cold. Some viruses have even been implicated in the development of
cancer.

We also have several positive relationships with microorganisms. Some soil bacteria fix
atmospheric nitrogen, making it available to the plants we eat. Certain fungi grow symbiotically
with plant roots, increasing their ability to obtain food and moisture from the soil. Others fungi
are themselves quite tasty. Bacteria and protests that live in our intestines help us gain nutrition
from food.

Microorganisms have also gained importance as tools in the scientific world. Since most have
simple life cycles and can reproduce rapidly, they make ideal model organisms. Topics like
genetics that are difficult to study in larger organisms because of the time and expense involved
in raising them can easily be studied in microorganisms living in Petri dishes by the billions. As
gene therapy gains importance for the study and treatment of disease, characterizing viruses that
may be used to transport genes is becoming a priority for some scientists.

15
Overall, microorganisms are some of the most important living creatures. Their roles as
producers and recyclers make them vital in most ecosystems. A greater understanding of these
tiny creatures is vital for the study and preservation of our natural environment.

1.2 Types of Microorganisms

Microorganisms include bacteria, viruses, protozoa, microscopic fungi and algae.

1.2.1 Bacteria:

They are prokaryotic, unicellular microorganisms which lack chlorophyll pigments.

1.2.2 Viruses:

Viruses are ultra-microscopic, non-cellular living particles, composed solely of a nucleic acid
(DNA or RNA) core, surrounded by a protein envelope called capsid.

1.2.3 Protozoa:

They are eukaryotic, unicellular microorganisms which lack cell wall.

1.2.4 Fungi:

They are eukaryotic, heterotrophic microorganisms that fail to show any cellular differentiation
into true tissues like root, stem or leaf and in which vascular system is absent.

1.2.5 Algae:

They are eukaryotic, autotrophic microorganisms that fail to show any cellular differentiation
into true tissues like root, stem or leaf and in which vascular system is absent.

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1.3 Importance of Microorganisms

Microorganisms are present in almost all places on the earth. Despite their minute size, they have
tremendous importance in the maintenance of life on the earth. Due to the difference in the
activities of different microbes, they influence life in different ways.

Some of them are very helpful, for example, those which fix atm. nitrogen into biologically
useful forms, those which help in curd formation, those which recycle dead materials by
degrading them into simpler substances and those which help in preparation of wine.

Some other microbes are very harmful in that they cause diseases in plants and animals, spoil
food or raw materials of food kept in normal conditions as well as they degrade rubber, paints,
textiles, metals and even insulations on electric wires.

Therefore, it is essential to study these microbes in details, so that by various techniques their
harmful effects can be overcome and beneficial effects be utilized for enhancing quality of life
on the earth.

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1.4 function of microorganisms?

The involvement of invisible organisms in many diseases in humans was suspected as early as
the 13th century by roger bacon .In the 16th century, Girolamo Fracastoro of virona suggested
that many human diseases where transmitted from person to person by small creatures. This was
also indicated by kircher in 1658.in 1762 Vonplenciz of Vienna suggested that different
invisible organisms were responsible for differet diseases. Theodore Schwan (1837) and Herman
Helmholyz (1843) proposed that putrefaction and fermention where connected with the presence
of the organisms derived from air. Finally, pasteeur in 1875 showed that wine fermentation from
grapes and souring of wine were caused by microorganisms. He also proved that spoilage of
meat and milk was associated with growth of microorganisms.

Later he showed the association of microorganisms with several diseases in humans, cattle,
sheep and he also developed vaccines against a few human and animal diseases caused by
microorganisms including rabies. Robert Koch in Germany (in the 1880s and 1890s), isolated
pure culture of bacteria responsible for anthrax, cholera, TB. He also developed the famous
Kochs postulates to associate a specific bacterium as a causative agent for a specific disease.
Along with associates he also developed techniques of agar planting methods to isolate bacteria
in pure cultures and to determine microbial numbers in a sample, the petri dish(by petri in his
laboratory), staining methods for better microscopic observation of bacteria, and use of steam to
sterilize materials to grow bacteria.

With time, the importance of microorganisms in human and animal diseases, soil fertility, plant
diseases, fermentation, food spoilage and food borne diseases and other areas was recognized,
and microbiology was developed as a specific discipline. Later, it was divided into several sub
disciplines, such as medical microbiology, soil microbiology, plant pathology and food
microbiology.

MICROORGANISM INVOLVE IN FOOD PRODUCTION

Fermentation:

C2H12O6 2C2H5OH + 2CO2 + energy

Dairy Products: cheese, yogurt and butter

Wine and bread: yeast (Saccharomyces cerevisaea)

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1.5 Food Microbiology

Food microbiology is the study of microorganism that inhabits, create, or contaminate including
the study of microorganisms causing food spoilage.

Current status

In the early 20th century, studies were conducted to understand the association and importance
of microorganisms, especially pathogenic bacteria in food. Specific methods were developed for
their isolation and identification. The importance of sanitation in the handling of food to reduce
contamination by microorganisms was recognized. Specific methods were studied to prevent
growth as well as to destroy the spoilage and pathogenic bacteria.

There was also some interest to isolate some beneficial bacteria associated with food
fermentation, especially dairy fermentation, and study their characteristics. However after the
1950s, food microbiology has entered a new erra. Availability of basic information on the
physiological, biochemical, and biological characteristics of diverse types of food, microbial
interactions in food environments and microbial physiology, biochemistry, genetics, and
immunology has helped open new frontiers in food microbiology (Ray, 2003).

1.7 Development of early food Microbiology

It is logical to comprehend that early homo ancestor, the hunters and gatherers were aware of
food spoilage and food borne diseases. Even without any perception of the causative agents, they
used ice and fire to preserve foods and make them safe. Around 8000 B.C., as agriculture and
animal husbandry were adopted by early civilizations, food supply, agriculture produce, became
available in abundance during the growing season. Preservation of food many food preservation
methods such as drying, cooking, backing, smoking, salting, sugaring(with honey), low
temperature storage (in ice), storage without air(in pits), fermentation(with fruits, grains and
milk), pickling, and spicing were used, probably mainly to reduce spoilage.

However, one cannot be sure whether the society at that time was recognized the implications of
diseases transmitted through food. In the later periods, however, the scriptural injunctions laid by
many religions suggest that the societies recognized an association of diseases with some foods.

Some of the revolutions such as not eating meat from a diseased animal or an animal killed by a
scavenger, or not eating the food that appeared unnatural or handled by an un clean person,
19
where developed to safeguard the health of citizens against food borne diseases. Fermentation
was used by many societies not only to preserve foods but also as a method to produce various
types of desirable foods from milk, meat, fish, eggs, grains, fruits, and vegetables.

20
Objectives:

To determine total aerobic count of different food samples

To obtain pure culture of bacteria from diverse food samples.

To identify various bacteria obtained from food samples on the basis of morphology and

biochemical tests.

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CHAPTER_02
REVIEW OF LITERATURE

Bacteria are the main and important cause of food spoilage. They thrive where food and
water are present and the temperature is suitable. To resist harm, some bacteria can form spores
tough reproductive cells that are able to survive under adverse conditions such as heat, cold and
chemicals. Thirty spoiled food samples were collected from Paonta sahib, India for isolation and
characterization of bacteria on the basis of morphology and biochemical reactions. Seven
bacterial isolates viz. Bacillus, Klebsiella, Pseudomonas, Escherichia coli, Lactobacillus,
Staphylococcus, and Micrococcus were found of which Bacillus, Klebsiella and Pseudomonas
were the dominating species in the spoilage of every category of food material. In fruits,
vegetables and meat Klebsiella was the most potent spoiling bacteria. In meat, milk and in fatty
products Pseudomonas was found in most of the spoilages. Although Klebsiella, Pseudomonas
and Bacillus were dominant in spoilages Escherichia coli, Staphylococcus and Micrococcus was
also recovered from considerable categories of the spoiled sample. The bacteria especially gram
negative were key responsible for food spoilage (Kumar et al., 2011).
Fresh fruits and vegetables promote good health but harbor a wide range of microbial
contaminants. Fifteen samples of fruits and vegetables were analyzed to assess the microbial
quality. Mean microbial load ranged from 1.3106 to 3.0107 cfu/ml. Staphylococcus aureus,
Klebsiella ssp., Salmonella spp. were the most frequently isolated bacteria while the
Actinomycetes and Escherichia coli were the least frequently isolated organisms. The effect of
washing by acetic acid and exposure time on microbial load of fruits and vegetables were also
assessed. Increasing concentration of vinegar from 0.5-2% decreased microbial population from
15-82%. Least microbial load for all vegetables were obtained when exposed to 2.5% vinegar
solution for 10 minutes (Eni et al., 2010).
Lactobacillus is a genus of lactic acid bacteria and described as heterogeneous group of
regular, gram-positive, rod shaped, non motile, non-spore forming bacteria with absence of
catalase enzyme (Holt et al., 1994). A study was conducted in Himachal Pradesh, India where 30
dairy samples were utilized for investigation of microflora. In total 12 bacteria isolates were
identified as Lactobacillus spp. after growth on MRS agar medium and confirmation by

22
morphological investigation, grams staining and specific biochemical tests (A. Kumar & D.
Kumar, 2014).
Gut microflora is considered very important for maintenance of human health. The
inherent growth of these microorganisms is also related to diet. Lactobacillus and bifidobacteria
are of prime importance in this respect. Milk and fermented dairy products are considered as
very good source of these microorganisms. Thus, an investigation was carried out to isolate and
characterize a potential probiotic bacteria from some commonly consumed food materials.
Consequently a bacterium identified as Lactobacillus casei was obtained from fermented dairy
product which showed potential as a probiotic owing to its antibiotic resistance, antimicrobial
potential and ability to survive under acidic environment (Mishra and Sharma, 2014).
The fermented maize starch known as Ogi in Yoruba and Akamu in Igbo is a popular
staple food. It is a traditional weaning food used mainly in West Africa. Bacteria were isolated
from steep water and aqueous Ogi. The bacteria assessment at the critical points of production of
Ogi was aimed at establishing the sources of contamination. The steep water and the aqueous
Ogi were screened for bacteria. The bacteria organisms were isolated from nutrient agar, cysteine
lactose- electrolyte-deficient (CLED) agar, de man, rogosa and sharpe (MRS) agar. The
leuconostoc specie was isolated from nutrient agar and Lacto bacillus species were isolated from
de man, Rogosa and sharpe (MRS) agar. The bacterial species isolated from cysteine-lactose
electrolyte- deficient (CLED) agar were Escherichia, Pseudomonas and Proteus species. The
critical points of contamination of ogi during production could be soaking in water, grinding and
sieving through muslin cloth. Appropriate safety measures and good manufacturing practices
could ensure quality ogi that would be free from all these microbial contaminants (Onyeze et al.,
2013).
A study was carried out assessing the microbial colonizers of apple fruits sold in Owerri
Nigeria to determine its safety for consumption. Apple fruits are dependable source of vitamins,
it is rich in fiber, electrolytes, minerals and antioxidants and it is usually eaten fresh and raw,
making the vitamins fully available for the body. The fruits were washed-out separately in 10 ml
sterile distilled water to obtain suspensions which were assayed for total aerobic plate count,
coliform count, and fungal count and for specific pathogens. A count of 3.4105-4.5107
cfu/mL was obtained for Total aerobic plate count, while total coliform and total fungal counts
ranges from 2.4104-2.2106 and 5.0102-3.6105 cfu/mL respectively. Twelve bacterial and
seven fungal species were isolated. The apple fruits sold in major busy spots in Owerri were
23
contaminated with Shigella spp, S. aureus, Salmonella and B. cereus which are known pathogens
which indicated poor hygienic quality of fruits (Oranusi and Wesley, 2012).
Evaluation of microorganisms associated with deterioration of tomato and pawpaw fruits
was carried out. Analysis revealed that various bacteria and fungi in high densities participated in
the spoilage processes. Microbial species encountered were bacteria such as Micrococcus
varians, Lactobacillus fermenti, Bacillus subtilis, Rothia sp., Pseudomonas stutzeri, Leuconostoc
species. Fruits were washed with chlorinated water treatment of fruits with antimicrobial agents,
as well as some forms of refrigeration are necessary to increase shelf-life and reduce the risks of
microbial toxins which are deleterious to human health. (Chinedu & Emmanuel, 2014)
A study was conducted for determination of coliforms in drinking water samples from
different water sources in Hazara Division (Mansehra, Abbottabad and Haripur). 15 samples
(16.66%) from Mansehra, 18 samples (20%) from Abbottabad and 16 samples (17.77%) from
Haripur were found pathogenic respectively. Four different bacterial species were isolated
namely E.coli, P. aeruginosa, Salmonella and H. pylori while E coli was mostly isolated specie
that showed contamination of water sources with feceal matter (Humayun et al., 2015).

The investigation was designed on the microbial status of some crude herbal materials.
Different types of spices and medicinal plants were collected. Ten different fungal genera and 16
species were isolated and identified as Alternaria alternata, Aspergillus spp., Gliocladium sp.,
Hyalodendron diddeus, Memmoniella sp., Penicillium spp., Rhizopus spp., Syncephalastrum sp.,
Cladosporium lignicolum and Ulocladium botrytis. The total number of isolated fungi from the
all sixteen selected samples was serially diluted and plated on Potato Dextrose Agar (PDA)
medium was (20310P3P) cfu/g. samples. Aspergillus spp. and Penicillium spp. were more
frequently detected, while Stachybotrys sp., Syncephalastrum racemocum, Uocladium botrytis,
Alternaria alternata, Cladosporium lignicolum and Gliocladium catenulatum were less frequently
detected.
The study were investigated on the spoilage fruit fungi and their plant cell wall
degrading enzymes of various fresh postharvest fruits sold in Jeddah city. Ten fruit spoilage
fungi were isolated and identified as follows Fusarium oxysporum (banana and grape),
Aspergillus japonicus (pokhara and apricot), Aspergillus oryzae (orange), Aspergillus awamori
(lemon), Aspergillus phoenicis (tomato), Asper(peach), Aspergillus niger (apple), Aspergillus
flavus (mango), Aspergil gillus tubingensis lus foetidus (kiwi) and Rhizopus stolonifer (date).
The plant cell wall degrading enzymes xylanase, polygalacturonase, cellulase and -amylase were
screened in the cell-free broth of all tested fungi cultured on their fruit peels and potato dextrose

24
broth (PDB) as media. Xylanase and polygalacturonase had the highest level contents as
compared to the cellulase and amylase. Aspergillus spp. are widespread and the fungal
polygalacturonases and xylanses are the main enzymes responsible for the spoilage of fruit
Cereal and its products can be contaminated with fungi in the field, The aim of study was
the isolation and identification of some fungi associated with four kinds of Libyan food products
of different trademarks. Twenty four (24) samples of couscous, macaroni, wheat flour and rice
regularly used for human consumption by Libyan family were collected from local markets in
the city of Alzawia, Libya. The results reveal isolation of 113 isolates belonging to nine genera:
Penicillium, Aspergillus, Fusarium, Paecilomyces, Alternaria, Rhizopus, Mucor, Scopulariopsis
and Cladosporum. , several of which are known as main producer of mycotoxins especially A.
flavus which are known to produce aflatoxins, Aspergillus niger, Aspergillus carbonarious,
Penicillium chrysogenum and Penicillium verrucosum known to produce ochratoxin and
Fusarium oxysporum and Fusarium chlamydosporum known to produce fumonisins and
trichothecenes.
A survey were conducted to assess the extent of loss in mangoes at wholesale and
consumer levels caused by fungal spoilage during post-harvest. Mango fruits were purchased
from different markets in Saudi Arabia, and the degree of losses due to fungal spoilage was
assessed at the different levels of marketing. Fungal spoilage was found to be the highest at the
consumer level and least at the wholesale level. Aspergillus flavus rot, Aspergillus niger rot, and
Penicillium spp. rot were the commonest diseases affecting the mango fruit
A study were conducted in which medium were developed which permitted isolation,
apparently for the first time, of the bacteria responsible for the acid production in the 100-year-
old San Francisco sour dough French bread process. Some of the essential ingredients of this
medium included a specific requirement for maltose at a high level, The bacteria were
grampositive, nonmotile, catalase-negative, heterofermentative lactobacilli.

25
Chapter-03

MATERIALS AND METHOD

3.1 Materials

Following chemicals and media will be used for this research.

1. Distilled water
2. Nutrient Agar
3. Butterfield phosphate buffer
4. Crystal violet stain (1%)
5. Lugol iodine solution
6. Safranin
7. Acetone
8. Hydrogen peroxide (3%)
9. Glucose phosphate broth
10. Methyl red indicator
11. Simmons citrate medium
12. Oxidase reagent
13. Tryptone water
14. Kovacs Reagent

3.2. Equipment

Following equipment will be used for this research.

Incubator (Binder 14D-78532)

Autoclave (JSAC-60)
Microscope (nuna WETZLAR)
Refrigerator (Dawlance)
Weighing balance (GF-6100)
Safety cabinet (TELSTAR AV-100)
Colony counter (Bhujhar-2003)
Blender (Waring)
26
3.3 .Sample Collection

Food Samples of fruits, vegetables, dairy, based products were collected from PARC display
shop and local market of chakshehzad. These samples were processed at FSPDI, NARC labs to
calculate microbial load, isolation and characterization of microorganisms. The sample names
were coded for simplicity on the basis of their types. The codes with their name are listed in table
1

3.4 Bacteriological Analysis: Bacteriological analysis was done by selective media method by
Presscott, 2002 and Sherman, 2005.

3.5 Morphological Characterization The isolated microbes were characterized on the basis of
simple staining and gram staining (Holt et al., 1994; Sherman, 2005).

3.6. Biochemical Characterization The isolates were characterized by biochemical tests viz.
IMViC reactions i.e. indole test, Methyl Red test, Voges Proskauer test and Citrate utilization
test, Nitrate Reduction.

S.NO SAMPLE SAMPLE CODE ISOLATE CODE

TYPE

1 Apple S1, I1

2 Meat S2 I2

3 Tomato S3 I3

4 Yougourt S4 I4

27
28
3.7 Media Preparation

Nutrient Agar will weigh and dissolved in distilled water according to manufacturers
instructions then it will be autoclaved at 121C for 15 minutes prior to use

3.7.1 Sample Preparation:

i. 25g portion of each food sample were blended in Waring blender at 10000-12000 rpm for
1 minute and then volume will be made up with 225 ml butterfield phosphate buffer
ii. 1ml of each homogenized food sample were added into the test tube containing 9ml of
butterfield phosphate buffer which will give us 10-1 dilution.
-1
iii. Serial dilutions will be performed from 10 to 10-3 by taking 1ml volume from 10-1
dilution and mixing it in 9ml buffer solution and then again taking 1ml volume from 10 -2
dilution and dissolving it in 9 ml buffer to produce 10-3 dilution.

29
30
3.7.2 Total viable count:

i. Each plate were labeled on top for identification of food sample, dilution factor, date

and name of analyst.

ii. One milliliter of each food sample dilution from 10-1 to 10-3 was pipette into nutrient

agar plates.

iii. Warm media (45-46C) were poured into Petri dishes containing food sample

dilutions and then allowe to cool and solidify.

iv. Gentle shaking of these plates will be done after the pouring agar for uniform

distribution of microorganisms from food samples.

v. The Petri plates were placed in incubator at 37C for 2-5 days in upside position after

the solidification of media to prevent condensation of water vapors.

vi. Colonies in nutrient agar plates were calculated with colony counter. The number of

colony forming units (CFU) per food sample will be reported after consideration of

dilution factor (FAO, 1997).

3.7.3 . PURIFICATION OF BACTERIAL ISOLATES

Petri plates were check for microorganisms growth after 2-5 days.
Different colonies of microorganisms were selected and their streaking was done on
nutrient agar under aseptic conditions with sterile wire loop to obtain pure culture
isolates.
The plates were incubated aerobically at 370C for 24 hours (Kiiyukia, C. 2003).

3.7.4 IDENTIFICATION OF VARIOUS ISOLATES OBTAINED IN THE CULTURES

Microscopic examination, gram staining and following biochemical tests will be carried out for

the characterization and identification of the organisms.

i. Catalase test

31
 ii. Citrate utilization test

iii. Indole test

iv. Oxidase test

v. Methyl red test

3.8 Grams Staining

It is most widely practiced method for differentiation of gram negative and gram positive

bacteria on the basis of their cell wall characteristics by checking their affinity to specific

combination of staining reagents. The tests will be performed in accordance with procedure

described by (Awan and Rehman, 2005).

Place a drop of water on clean glass slide. Now using aseptic technique transfer tiny amount of

microbial material from culture plates to glass slide by gentle stroke of sterile wire loop. Spread

this microbial substance softly on slide with drop of water through wire loop. Allow the slides to

air dry. Heat fix the smear on slide by passing it over a Bunsen flame thrice.

Apply liberally crystal violet stain (1%) on bacterial smear and leave for 60 seconds. Wash the

slide with distilled water. Now flood the smear with Lugols iodine solution (mordant) and leave

for 60 seconds. Rinse iodine with water and decolorize the slide with acetone for 2-3 seconds.

Rinse the slide with water and counter stain with safranin for 60 seconds and wash off again.

Blot dry the slide and examine with the oil immersion 100X lens. A purple colour signifies Gram

(+) positive bacteria while the colour of the safranin which is red signifies Gram (-) Negative

bacteria.

32
3.8.1 Catalase Test

This test will be done according to procedure described by Cheesbrough (2006). The test will be

performed by dropping and mixing loopful of the isolate hydrogen peroxide (3%) on the slide.

The production of gas bubbles (O2) from the mixture which will occur almost immediately is a

positive reaction.

2H202 2H20 + 02

3.8.2 Methyl Red Test

This test is used to detect which of the bacterial isolates could produce and maintain sufficient

and stable acid production from glucose fermentation. The test is usually used as an aid in the

identification and differentiation of the Enterobacteriaceae. This test was performed according

to Cheesbrough (2006). Inoculate the suspected organism into a sterile buffered glucose-

phosphate broth and incubate at 37C for 24 hours. After 24 hours add five drops of methyl red

indicator, shake the mixture and observe. Appearance of bright red colour is positive result.

3.8.3 Citrate Utilization Test

This test was done according to Cheesebrough (2006). The test is used to identify which of the

isolates can utilize citrate as the sole sources of carbon for metabolism. The test is also used as

an aid in the differentiation of organisms in the Enterobacteriacea group. Inoculate simmons

citrate medium in sterile test tubes with a loopful of culture.

Incubate tube at 370c for 24 hours. A colour change from green to blue is a positive result. The

absence of any growth as well as no change in the colour indicates a negative reaction.

33
3.8.4 Oxidase Test

This test is used to identify microorganisms containing the enzyme cytochrome oxidase. This test

is done by dropping 2 5 drops of a freshly prepared oxidase reagent on a filter paper, the

suspected organisms are picked using a sterile wire loop and mix with the oxidase reagent. A

change from the normal colour to deep purple means a positive result, while no change means

negative (Cheesebrough, 2006).

3.8.5 Indole Test

This test is done according to (Cheesebrough, 2006) to distinguish among members of

enterobacteriacea. Escherichia coli and only some shigella strains are indole positive. The test

organism was inoculated in a test tube containing 3ml of sterile tryptone water at 37 oc for 24hrs.

After incubation 0.5ml of kovacs reagent is added and mixture is shaken gently. Appearance of

red colour on the surface of the layer within 10 minutes means positive, while no colour change

means negative

34
CHAPTER 4

RESULT AND DISCUSSION

Bacteria can cause fruits and vegetables to get spongy or slippery, or meat to develop a bad
stench. There are different spoilage bacteria which grow well at room temperature. The large
number of microorganisms and their waste products cause the unpleasant changes in odor, taste
and texture. In the present study different samples of spoiled food were collected from the
market of chaksehzad, Mehraban house Islamabad.

Morphological Characterization: The four isolates were characterized on the basis of colony
morphology and the staining characteristics. It was observed that 03 isolates were gram (-ve)
rods, and 01 isolate are gram positive (Fig 01)

35
 Table 4.1Morphological Characterization

S.N SAMPLE ISOLATE COLONY SIMPLE GRAM SUSPECT

O CODE CODE STAINING STAINING

01 S1 S1i1 Waxy Rods negative Klebsilla,E.coli,bas

growth illus

02 S2 S2i2 Thin Rods Gram Pseudomonas,basil

white negative lus
growth

03 S3 S3i3 Abundant, Rods negative Klebsilla,E.coli,bas

golden illus
growth

04 S4 S4i4 Cremy,wh Rods Gram Lactobasilus

itish positive
convex
colonies

36
Biochemical Characterization :These four isolates were characterized on the basis of
biochemical tests (Table 3). The tests performed to characterize the isolates were Indole,
Oxidase, , CU,NR, metylated red,catalase

37
 Table 4.2biochemical characterization of isolate

ISOLATE IDENTIFIED
INDOLE NR CU LACTOSE
CODE BACTERIA

S1i1 _ + _ _ Klebsilla,bascillus,E.coli

S2i2 _ + + _ Pseudomonas sp
klebsilla.E.coli,basillus

S3i3 _ + + A Klebsilla,basillus,E.coli

S4i4 - + - + Lactobasillus sp.

38
In this revise the four samples of various categories of food such as fruit, vegetable, meat, milk
and milk products, were collected from the local market of islamabad. these isolates were
characterized on the basis of biochemical identification The result obtained from the data shows
that the bacteria found in spoiled samples was Bacillus, Klebsiella, Pseudomonas, E.coli,
Lactobacillus, and there prevalence was 18%, 15%, 14%, 10%, 7%, 5%, 4% respectively (Fig.
3)pneumonia is a potent enteroinvasive food borne pathogen and causes serious illness (Sabota et
al., 1998

In order to understand the prevalence of bacteria for different categories of food The bacteria
found to spoil the fruits were identified as Klebsiella,Basillus , E.coli, Pseudomonas,
Lactobacillus and there prevalence was 31.25%, 25%,18.75%, 6.25% and 6.25% respectively
(Fig. 4). A Bacillus strain was isolated from spoiled apple juice. This strain was acidophilic with
a growth range between pH 2.5 and 5.5. This organism could be a threat to fruit juices during
storage at temperatures greater than or equal to 26C because its spores were able to survive
pasteurization condition (Cerny et al., 1984). Lactobacillus fermentum and Lactobacillus
plantarum are the heat resistant lactic acid bacteria obtained from spoiled acidified fruits
(Shearer et al., 2002).

Among the vegetable tomato samples was collected and two isolates were recovered from them.
The bacteria found to spoil the vegetables were identified as Klebsiella, Bacillus, E.coli,
Pseudomonas and there prevalence in vegetable sample was found to be 33.33%, 23.80%,
14.28%, 14.28%, 14.28% respectively (Fig. 5). Pseudomona and Klebsiella are responsible for
spoilage even in frozen vegetables (Manani et al., 2006).

Among the meat products three isolates were recovered from the meat sample. The bacteria
found to spoil the meat were identified as Pseudomonas, Klebsiella, Bacillus, E. coli, (Fig. 6)
prevalence of the suspect in meat sample was found to be 38.46%, 23.07%, 23.07%,
7.69%,respectively (Olajuyigbe et al., 2006).

Among the milk and milk products 2 isolate were recovered from the sample.. The bacteria
found to spoil the milk were identified as Lactobacillus, Pseudomonas, Bacillus, and there
prevalence in milk samples was found to be 44.44%, 22.22%, 22.22%, 11.11% respectively (Fig.
7). Mesophilic Lactobacillus sp. is the dominant organisms in mature Cheddar cheese (Jordan et

39
al., 2002). Species of the genus Lactobacillus are widespread in nature and can be found on
plants or material of plant origin, in manure and man-made habitats such as sewage, in
fermenting or spoiling food and also in association with the intestinal tracts and mucous
membranes of man and many animals (Chenoll et al., 2005). Lactobacilli from breast milk
could contribute to an anti-infective protection in neonates and would be excellent candidates for
the development of infant probiotic products (Olivares et al., 2006). Degradation of milk
components through various enzymatic activities associated with the contamination of dairy
products by Pseudomonas sp can reduce the shelf life of processed milk (Cormier et al., 1991;
Belgin et al., 2006).

40
Fig.2 gram bacteria in food samples on basis of staining

12

10

0
Gram Negative Rod gram positive rod

41
Fig.3 prevalance of bacillus, klebsiella, pseudomonas,E.coli, Lactobacillus

20

18

16

14

12

10

0
bacillus klebsiella pseudomonas E.coli lactobacillus

42
 Fig4.prevelence of bacteria in fruit sample

35

30

25

20

15

10

0
klebsilla Bacillus E.coli pseudomonas lactobacillus

43
 Fig 5.prevelance of bacteria in vegitables

45

40

35

30

25

20

15

10

0
klebsiella bacillus E.Coli pseudomonas

44
 Fig6.prevalence of bacteria in meat samples

45

40

35

30

25

20

15

10

0
pseudomonas klebsiella bacillus E,coli

45
 Fig.7 prevalance of bacteria in milk product

50

45

40

35

30

25

20

15

10

46
Conclusion
From the present study it was concluded that five bacterial isolates viz. Bacillus, Klebsiella,
Pseudomonas, E.coli, Lactobacillus, was isolated from the spoiled food samples. It was found
that the Bacillus, Klebsiella and Pseudomonas were the dominating species in the spoilage of
every categories of food material. In fruits, vegetables and meat Klebsiella was the most potent
spoiling bacteria. In meat, milk and in fatty products Pseudomonas was found in most of the
spoilages. Although Klebsiella, Pseudomonas and Bacillus were dominant in spoilages E.coli,
was also recovered from considerable categories of the spoiled sample.(fig;8) Lactobacillus was
recovered from the acidified spoilages as well as from the milk spoilages. The strains of
Lactobacillus in fresh milk act as probiotic but in spoilages it is responsible for the acid
production and the sour taste and rancidity.

47
bacteria in food samples

bascilus
klebsiella
pseudomnas
ecoli
lactobacillus

48
45
40
35
30
25
meat
20
15 fruit
10 milk
vege
5 vege
0 milk
fruit
meat

49
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