INTRODUCTORY

MICROBIOLOGY

BY
USMAN, A O. (MRS) & ISIAKU,
H, Y (MR.)

I
 DEDICATION

This book “Introductory Microbiology” is dedicated to

Almighty Allah.

PREFACE
II
This book “Introduction to Microbiology” is a work of expert
microbiologists with vast experiences and dynamic skills in the
teaching of microbiology who aimed at assisting students of
microbiology in colleges, polytechnics and universities in
broadening their knowledge of microbiology. This book
contains all the basic knowledge that students need to have
about microbiology

This write-up covers most aspects of microbiology which are

well discoursed and logically treated with explicit examples
which include: scope of microbiology, branches of
microbiology, development of microscope, roles of scientists in
development of microscope, roles of microbiology in phases of
life, principles of microscopy, types of microscopes, staining
techniques, characteristics of microorganisms, microbial
growth, factors influencing microbial growth and host of other
relevant topics.

The book deals with topics according to the guidelines of the

new National Board Technical Education (NBTE) Curriculum.
The book is therefore valuable, useful and helpful to students
in the colleges, polytechnics and universities.

ACKNOWLEDGMENT

III
Our utmost and profound gratitude goes to Allah for His grace
and mercy on us for giving us the strength and wisdom to write
this book.

We wish to express our sincere appreciation to the Provost and

Management of the School Health Technology, Offa, Kwara
State, for providing an enabling academic atmosphere for this
type of book to be written.

We also appreciate the love, support and encouragement of

both the Head of dispensing optictionary (DOP) and the
Coordinator of Unit of Basic Science; Mallam Zikirullahi
Oluwe who have been highly encouraging.

Our deepest gratitude goes to all our colleagues and all the staff
and also to a large number of students whose interests,
criticisms, perpetual questions and encouragement have made
this book possible. We are also grateful to all our friends too
numerous to mention.

We sincerely appreciate the moral support and understanding

received from beloved spouse and children. We are also
indebted to a number of people for their help, prayer and
advice and also to those authors whose books were consulted at
various stages in the preparation of this book.

To you all, I say God bless you.

FORWARD

IV
This book; “Introductory Microbiology” has been put together
by a seasoned and experienced biology and microbiology
lecturers. The book will serve as a quick reference for all
students of biology, microbiology, parasitology, zoology,
science laboratory technology and any student taking a course
in microbiology and physiology. The field of microbiology is
quite vast and this book has managed to present all the main
principles in a summarized and concise form. It presents the
fundamentals of microbiology in a manner comprehensible to
students with a minimal background in microbiology. It is my
sincere hope that this book will be interesting and valuable to
all students and teachers of microbiology. The authors
welcome your comments and suggestions.

Usman, K.M.
Senior Lecturer
Department of Biological Sciences
Federal Polytechnic, Offa,
Offa, Kwara State

TABLE OF CONTENT

V
Title page i
Dedication ii
Preface
iii
Acknowledgment iv
Forward v
Table of content vi
CHAPTER ONE
1.0 History and scope of microbiology 1
1.1 Scope of microbiology 1
1.2 Branches of microbiology 1
1.3 Development of microscope and microbiology 1
1.4 The scientists involved in the development of
microscope and microbiology 2
1.5 The role of microbiology 4
CHAPTER TWO
2.0 Principle of microscopy 8
2.1 Types of microscope 8
2.2 Microbial staining techniques 13
2.2.1 Gram’s staining 14
2.2.2 Spore staining
15
2.2.3 Flagella staining 15
2.3 Prokaryotic and eukaryotic cells 16
CHAPTER THREE
3.0 Systemic microbiology 18
3.1 Characteristics of microorganism 18
3.2 Morphological characteristics 19
3.3 Biochemical characteristics of classifying
microorganisms 21
CHAPTER FOUR
4.0 Growth of microorganisms 22
4.1 Factors influencing microbial growth 22
4.2 Microbial growth 25
VI
CHAPTER FIVE
5.0 Isolation, cultivation and preservation of
microorganisms 28
5.1 Culture media 28
5.2 Preparation of culture media 31
5.3 Sterilization of culture media 32
5.4 Types of culture 33
5.5 Isolation of pure culture 33
5.6 Maintenance of pure culture 35
CHAPTER SIX
6.0 Control of microorganisms 37
6.1 Reasons for controlling microorganisms 37
6.2 Terminologies used in controlling microorganism 37
6.3 Types of sterilization and disinfection 39
References 40

VII
 CHAPTER ONE

1.0 HISTORY AND SCOPE OF MICROBIOLOGY

1.1 SCOPE OF MICROBIOLOGY

The word Microbiology is from the Greek words ‘MICRO’

which means small or tiny, ‘BIOS’ which means life and
‘LOGOS’ which means study of description. Therefore
Microbiology can be defined as the scientific study of
microorganisms. Microorganisms are those living organisms
that are too small to be seen clearly and studied as individuals
by the unaided human eyes, such living things are said to be
microscopic i.e. organisms that cannot be seen with the naked
eye unless with the aid of microscope.

Microbiology is an applied science with significant

implications in genetics, biochemistry, food science, ecology,
immunology, agriculture, medicine and a variety of other
fields.

1.2 BRANCHES OF MICROBIOLOGY

Microbiology as a broad spectrum discipline has specific in its
areas of specialties. These various specifics are simply referred
to as branches of microbiology. These branches include
Bacteriology, Immunology, Mycology, Phycology/Algology,
Protozoology and Virology and so on.

1.3 DEVELOPMENT OF MICROSCOPE AND

MICROBIOLOGY
Origin: The origin of the word microscope according to the
Online Etymology Dictionary is as follows: Microscope was
coined from the word “microscopium” "an instrument for
viewing what is small," from Greek. micro + skopion. "Means

1
of viewing," from skopein "look at." Microscopic "of minute
size"

The discoverer of the microbial world was a Dutch merchant

named Anthony Van Leeuwenhoek (1632- 1723). He carved
out a niche for himself in microbial studies which later gained
the attentions of several scientists. His early discoveries were
purely based on honest imagination and he was inquisitive as
well as curios and studied any objects with his invented
microscope.

To the end, Leeuwenhoek’s placed in the scientific history

depended not so much on his skill as a microscope maker but
essentially on extraordinary range a skill of his microscopic
observations. His inquisitive tendencies led him to examine a
wide variety of material he encountered-rivers, well and sea
water as well as vinegar and pepper. He equally observed
structure of seeds, embryo of plants and small invertebrate
animals. He extended his discoveries to existence of
spermatozoa and red blood cells. As an erudite intellectual his
scientific ingenious rests on the discovery of the microbial
world, the animalcules or small animals as called by his
contemporaries. All the unicellular microorganisms known
today- algae, bacteria, yeasts and protozoans were first
discovered by Leeuwenhoek. He also emphasized the
abundance of microorganisms.

1.4 THE SCIENTISTS INVOLVED IN THE

DEVELOPMENT OF MICROSCOPE AND
MICROBIOLOGY

14th century: spectacles first made in Italy

2
1590: Two Dutch spectacle-makers and father-and-son
team, Hans and Zacharias Janssen, create the first
microscope.

1667: Robert Hooke's famous "Micrographia" is published,

which outlines Hooke's various studies using the microscope.

1675: Anton van Leeuwenhoek, who used a microscope with

one lens to observe insects and other specimen. Leeuwenhoek
was the first to observe bacteria. 18th century: As technology
improved, microscopy became more popular among scientists.
Part of this was due to the discovery that combining two types
of glass reduced the chromatic effect.

1830: Joseph Jackson Lister discovers that using weak lenses

together at various distances provided clear magnification.

1878: A mathematical theory linking resolution to light

wavelength is invented by Ernst Abbe.

1903: Richard Zsigmondy invents the ultra-microscope,

which allows for observation of specimens below the
wavelength of light.

1932: Transparent biological materials are studied for the first

time using Frits Xernike's invention of the phase-contrast
microscope

1938: Just six years after the invention of the phase contrast

microscope comes the electron microscope, developed
by Ernst Ruska, who realized that using electrons in
microscopy enhanced resolution.

3
1981: 3-D specimen images possible with the invention of the
scanning tunneling microscope by Gerd Binnig and Heinrich
Rohrer.

1.5 THE ROLE OF MICROBIOLOGY

The role of microbiology in nature cannot be underestimated,
microbiology play very vital role in waste disposal, industries
(food and diary, alcohol and beverages). Chemical industry,
biological warfare, agriculture, medicine just to mention but a
few. The role of microbiology is so versatile that it touches
either directly or indirectly all forms of life. In fact, the
economy of nature would not have been completed without the
critical role of microorganisms in the recycling of mineral
elements. Generally, microbiology deals with microorganisms
or microbial techniques in commercial enterprises.

Microbiologist through the knowledge of microbiology can

identify, isolate, diagnose, prevent pathogenic microorganism.
They can also play a major role in various biochemical
processes such as biodegradation, bio-deterioration, climate
change, food spoilage, epidemiology and biotechnology.

WASTE /SEWAGE TREATMENT

Wastes constitute ‘leftovers’ which are directly and indirectly
discharged into the bodies of water. Most waters for
consumption received organic and inorganic wastes usually
inform of industrial waste, thus constituting health hazards.
Microorganisms are most depended upon for the
decomposition of these offensive organic matters and their
reduction to soluble and useful materials.

Cleaning of oil spillage

Crude oil spillage causes a lot severe effects on both the
vegetation cover and the lives of animals including
4
microorganisms inhabiting the soil. The oil consuming bacteria
can therefore be used to clean oil spillage that causes a lot of
environmental and health hazards. Such bacteria attack and
destroy petroleum wastes to virtually non-toxic or non-
hazardous product.

Single Cell Proteins (SCP)

Another important aspect of industrial microbiology is the
development of single cell protein (SCP). Single cell proteins
are made from bacteria that are non-pathogenic and are capable
of feeding on many agricultural waste materials that contain a
lot of cellulose. Their ability to multiply quickly and increase
in great number couple with easy handling and manipulations
of microorganisms tends to make SCP an important alternative
for the conventional plant and animal proteins.

INDUSTRIES

Beverages and food industries

In alcoholic and beverages industries, the yeast,
Saccharomyces cerevisiae are normally used to convert sugars
into alcohol and carbon dioxide with little quantity of energy.
Alcohol tends to constitute the main product. This process is
essentially and anaerobic process that can be represented with
the following equation;

C6H12O6 2C2H5OH + 2CO2 + E

Glucose Alcohol Energy

This same yeast is also useful in the leavening of bread, thus

creating lattice through which the carbon dioxide releases tends
to escape when kneaded flour is subjected to the temperature
condition of the oven.

5
Yoghurt: is usually made from milk through that action of a
bacterium known as Lactobacillus acidophilus. L. acidophilus
produced lactic acid which sweeten the yoghurt.

Cheese: is also made from milk through the action of bacteria.

Different bacteria are used to produce different type of cheese.
Flavouring of cheese is also done through the action of
bacteria.

Butter: is made and flavored through the action of bacteria on

butter milk. Streptococcus cremoris is a typical example
known. Butter is the fat present in the milk of cow.

Vinegar: is made from fruit juice through the action of

bacteria. Acetobacter aceti is a vinegar bacterium. However,
commercial vinegar usually contains about 5% Acetobacter
aceti.

MEDICINE
Microorganisms have been seen to be involved in the various
stages in the production and processing of drugs. Some
microorganisms are used in the manufacture of hormones. For
instance, progesterone is converted to cortisone through the
action of a fungus Aspergillus spp. The blue mould Penicilium
notatum commonly seen on decaying citrus fruits produces
antibiotics penicillin which was initially regarded as ‘magic’
drug.

AGRICULTURE
The role of microbiology tends to command attention of
specialists. Among the branches of microbiology is plant
pathology, which deals with the studies of the causatives of
disease to plant and their control.

6
Nitrogen fixation: some very useful and indispensable
microorganisms that are commonly found in the soil are the
nitrogen fixers.

MICROBIAL INSECTICIDES
Microbiology and entomology tends to pose a scientific
challenge leading to the development of ‘biological
insecticides’. For instance, Bacillus thuringiensis produce
poisons that are very effective against certain insects but
harmless to other forms of life. Destructive ‘cabbage looper’
and a number of other pests are very susceptible to specific
viruses.

BIOLOGICAL WARFARE
This is another area of microbiological breakthrough which
involves strategies application and/or usage of biological
agents either to kill or incapacitate our enemies. Some viruses
are used as lethal biological agents such as laser viruses. This
particular virus tends to kill fast than one can ever imagine.
Therefore, lethal agents are usually avoided and incapacitated
one tends to be the one of choice. However, biological warfare
have received worldwide condemnation in strong terms,
nonetheless, the relative importance of this exercise

7
 CHAPTER TWO

2.0 PRINCIPLE OF MICROSCOPY

The principle of modern compound microscope is the same as
that discovered by a Dutch man spectacle maker – Zaccharias
Jensen about 1590 and a Dutch merchant Anthony Van
Leeuwenhoek simple microscope with single lens. The use of
lens now referred to as lens system, an eye piece or ocular is to
magnify an already enlarged image produced by a first lens
called an objective. The total magnification of an object
therefore, is a product of the modern objective lens and ocular
lens. For instance, the total magnification of an object whose
image is formed when viewed with the objective lens of x100
and ocular lens of x10 is x1000.

Microscopes are designated as either light microscopes or

electron microscopes. The former use visible light or
ultraviolet rays to illuminate specimens. They include bright
field/light microscope, dark field, phase-contrast, and
fluorescent microscope. Fluorescent microscopes use
ultraviolet radiations whose wavelengths are shorter than those
of visible light and are not directly perceptible to the human
eye. Electron microscopes use electron beams instead of light
rays, and magnets instead of lenses to observe sub-microscopic
particles.

2.1 TYPES OF MICROSCOPE

LIGHT MICROSCOPE/ BRIGHT FIELD MICROSCOPE

This instrument contains two lens systems for magnifying
specimens: the ocular lens in the eyepiece and the objective
lens located in the nose-piece. The specimen is illuminated by
a beam of tungsten/visible light focused on it by a sub-stage
lens called a condenser, and the result is that the specimen
8
appears dark against a bright background. A major limitation
of this system is the absence of contrast between the specimen
and the surrounding medium, which makes it difficult to
observe living cells. Therefore, most bright field observations
are performed on nonviable, stained preparations.

Revolving power of Microscope

The usefulness of a microscope depends not solely on the
degree of magnification, but on the ability of the microscope to
separate clearly objects that are very close together i.e. the
ability to revolve. The revolving power of a microscope is
defined as a maximum distance that can exist between two
9
points such that each point is observed as a separate entity.
Revolving power therefore, determines how much details of
the object in focus can be seen.

Immersion oil
Oil immersion lenses are specially designed for use with
immersion on the fluids having a refractive index like that of
glass. During operation, the immersion fluid is place on the
slide and should be removes from the lens after use or before
the instrument is stored. Such lens should be cleansed with lens
paper or tissue only. Avoid the use of fluid solvent as this
might cement the instrument while other materials may be
abrasive enough, thus scratching the lens.

Methods in Light Microscope

Hanging- Drop Techniques
Microorganisms discovered in their natural state are best
viewed when suspended in a clear fluid such as water, saline
solution of broth. Based on the simple fact that the cells are
transparent, colorless retractile and so tiny, they are difficult to
find and even identified in a drop of liquid or fluid e.g. cocci
which are only 1 or 2Fm in diameter.

During preparation, loopful of the culture of microorganism in

fluid medium is suspended in the center of a thin coverslip. On
each of the four corners are placed a tiny droplet of mineral oil
or petroleum wax or jelly. Then hold a hollow-ground slide,
depression side down, over the slip and bring the two into
contact. This is inverted quickly so as to prevent the run off to
one side. In addition there are tiny drops of clear mineral oil
run under the edges of the cover slip at each corner, it spreads
under the cover slip and prevents drying of the drop.

10
This technique enable individual to see the size, shape and
arrangement of microorganisms and their motion if motile. In
some cases, bright refractive granules and spores may be seen
within the cells. This technique is limited in scope but provides
valuable information. This is because cells of yeast, moulds,
protozoan and bacteria are colourless and transparent; hence,
there are a lot of difficulties in studying them.

Figure 2.1: Hanging –Drop Techniques

DARKFIELD MICROSCOPE
This is similar to the light microscope; however, the condenser
system is modified so that the specimen is not illuminated
directly. The condenser directs the light obliquely so that the
light is deflected or scattered from the specimen, which then
appears bright against a dark background. Living specimens
may be observed more readily with dark field than with bright
field/ light microscope.

PHASE-CONTRAST MICROSCOPE
Observation of microorganisms in an unstained state is
possible with this microscope. Its optics includes special
11
objectives and a condenser that make visible cellular
components that differ only slightly in their refractive indexes.
As light is transmitted through a specimen with a refractive
index different from that of the surrounding medium, a portion
of the light is refracted (bent) due to slight variations in density
and thickness of the cellular components. The special optics
convert the difference between transmitted light and refracted
rays, resulting in a significant variation in the intensity of light
and thereby producing a discernible image of the structure
under study. The image appears dark against a light
background. 

FLUORESCENT MICROSCOPE
This microscope is used most frequently to visualize specimens
that are chemically tagged with a fluorescent dye. The source
of illumination is an ultraviolet (UV) light obtained from a
high-pressure mercury lamp or hydrogen quartz lamp. The
ocular lens is fitted with a filter that permits the longer
ultraviolet wavelengths to pass, while the shorter wavelengths
are blocked or eliminated. Ultraviolet radiations are absorbed
by the fluorescent label and the energy is re-emitted in the form
of a different wavelength in the visible light range. The
fluorescent dyes absorb at wavelengths between 230 and 350
nanometers (nm) and emit orange, yellow, or greenish light.
This microscope is used primarily for the detection of antigen-
antibody reactions. Antibodies are conjugated with a
fluorescent dye that becomes excited in the presence of
ultraviolet light, and the fluorescent portion of the dye becomes
visible against a black background.

ELECTRON MICROSCOPE
In the electron microscope, the specimen is illuminated by a
beam of electrons rather than light, and the focusing is carried
out by electromagnets instead of a set of optics. These
12
components are sealed in a tube in which a complete vacuum is
established. Transmission electron microscopes require
specimens that are thinly prepared, fixed, and dehydrated for
the electron beam to pass freely through them. As the electrons
pass through the specimen, images are formed by directing the
electrons onto photographic film, thus making internal cellular
structures visible.

Scanning electron microscopes are used for visualizing

surface characteristics rather than intracellular structures. A
narrow beam of electrons scans back and forth, producing a
three-dimensional image as the electrons are reflected off the
specimen's surface.

2.2 MICROBIAL STAINING TECHNIQUES

Staining is the application of coloured substances called dyes
which tend to impart its colour on certain structure within the
cells against its background. The problem of observing living
transparent and motile organisms is overcome by the staining
techniques.
Microbial staining involves a procedure. Hence, in staining
procedure, a drop of the liquid containing the organism is
placed on a clean glass slide with a sterile wire loop and
allowed to air-dry (smear). The dry film is then fixed on the
slide, either with chemical fixatives or by passing over a blue
flame to coagulate the cell protein, appropriate stains are then
applied.

There are varieties of stains and staining procedures currently

is use, some stains are specific in that they will stain only a
particular structure of the cell, while others will stain only an
organism and no other organisms. Stains are generally
categorized into positive stain and negative stain. Positive
13
stains have strong attraction for one or more cell components
while negative stains cannot penetrate the cell components thus
making it highly visible by providing a contrasting dark
background.

TYPES OF STAINING TECHNIQUES

2.2.1 GRAM’S STAINING
A Danish scholar and physician named Dr. Hans Christian
Gram developed this staining technique while working in
morgue in Berlin in 1884. This technique classified bacteria
into two broad groups vis-à-vis Gram positive and Gram
Negative, hence referred to as differential stain. This technique
also revealed the size, shape and arrangement of the cells as
well as structural details.
Method
i. Make a thin smear of the cell suspension on a clean
glass slide
ii. Fix with chemical fixative or by passing the slide
containing the smear over blue flame
iii. Flood the slide with crystal violet for 30-60 seconds
iv. Flood with dilute solution of iodine which decreases the
solubility of purple dye forming-iodine complexes
v. Rinse off in gentle tap water
vi. Apply 95% alcohol which readily removes purple dye-
iodine complexes from some bacteria and not others.
vii. Flood with red counter stain such as congo red or
safranin for 30 -60 seconds
viii. Rinse off in gentle tap water
ix. Blot dry with blotting paper
x. Examine under oil immersion lens

14
2.2.2 SPORE STAINING
Spore formation is one of the protective devices by certain
bacteria to withstand certain unfavorable conditions in their
environment. Certain bacteria, mostly members of the genera
Bacillus and Clostridium produce endospores. A spore is
usually produced by bacteria cell, such spore may be terminal,
sub-terminal or central in their location.
Method
i. Make a thin smear of the bacterial suspension
ii. Heat fix
iii. Stain the smear with gram’s stain
iv. Blot dry with filter or blotting paper
v. Examine under oil immersion lens.

2.2.3 FLAGELLA STAINING

The bacteria flagellum is so tiny that it cannot be seen with
light or optical microscopes. However, by special staining
techniques, some stains can be made to deposit around the
flagellum so as to increase its diameter. Thus, making the
flagellum visible under the light or optical microscope.
Possession, number and arrangement of flagella are very useful
tools in bacteria classification.
Method
i. Gently was off some bacteria cells from slope or slant
using sterile water.
ii. Make a smear of thin drop on a clean glass slide
iii. Allow to air-dry (do not heat fix)
iv. Flood the slide with Leifson’s stain for 10 -15minutes
v. Rinse off gently in tap water
vi. Counter stain with methylene blue for 5 minutes
vii. Rinse gently in tap water
viii. Allow to air dry
ix. Examine under oil immersion lens
15
Leifson’s stain alone tends to stain flagella pink-red, while
with counter stains, the flagella stain red and the cells stain
blue.

2.3 PROKARYOTIC AND EUKARYOTIC CELLS

Prokaryotic cells
Prokaryotic cell (pro ‘before’ karyo ‘nucleus’) were probably
the first form of all life on earth. Their hereditary material i.e.
DNA is not enclosed within a nuclear envelope. There are non-
membrane-bound organelles within a prokaryotic cell. The
absence of a true nucleus occurs in two groups of organisms,
the bacteria and the blue-green algae.

Eukaryotic cells
Eukaryotic cells are more structurally complex than the
prokaryotic cells, it is also larger. The eukaryotic cell has a
16
distinct membrane bounded organelles are also present such as
mitochondria.

DIFFERENT BETWEEN PROKARYOTIC AND

EUKARYOTIC CELLS
PROKARYOTIC CELL EUKARYOTIC CELL
No membrane bound Membrane bounded
organelles organelles are present
No chromosomes (circular) Chromosomes present on
stands of DNA which DNA are located
No chloroplast only Chloroplast present in plants
photosynthetic lamella and algae.
Ribosome are smaller Ribosome are larger
No distinct nucleus, only A distinct membrane bound
diffuse area(s) of nucleus
Nucleoplasm with no nuclear
envelope
Flagella (if present) lack Flagella have 9+2 internal
internal 9+2 fibril fibril arrangement.
arrangement
No mitosis or meiosis occur Mitosis and/or meiosis
occur.
Example: Bacteria, Examples: Fungi, Protozoa,
Rickettsiae, Chlamydiae, Plants, Animals all other
Cynobacteria organism.

17
 CHAPTER THREE

3.0 SYSTEMATIC MICROBIOLOGY

Systematic microbiology is an important aspect of
microbiology that deals with the detailed study of individual
microorganism. This includes the study of morphology,
cultural characteristics, nutritional requirements, biochemical
characteristics, physiological properties, serology and
bacteriophage sensitiveness of various groups of
microorganisms. An important prerequisite for objective,
unbias systematic study of microorganism is to obtain and
maintain a pure culture in stock of microbes through series of
preliminary sub culturing; such pure cultures are usually
maintained on a well buffered agar slant.

3.1 Characteristics of Microorganisms

 Microorganisms are living organisms that cannot be seen
with naked eye. Hence, they are said to be microscopic.
 Microorganisms are said to be ubiquitous i.e. They are
found everywhere, in air, water, rivers, ponds, soil, food,
clothes, and on body surfaces.
 Most organisms are harmless and useful such as yeasts
(Sacchromyces cerevisiae) used in bread making and
fermentation of alcoholic beverages, some constitutes the
normal flora of the body. Others that are harmful include
Bacillus anthracis, Clostridium spp., Salmonella spp.,
Streptococcus spp., which are agents of various human
and animal diseases, food infection and food intoxication.
 They could be aerobic and anaerobic; Aerobic
microorganisms are those that depend solely on presence
oxygen for their metabolic activities while anaerobic
microorganisms do not depend on oxygen but rather on
carbon dioxide for their metabolic activities.

18
  Some microorganisms reproduce asexually by simple
division, spore formation, budding, fragmentation and
sexually by the formation and fusion of gametes.
 Some microorganisms are motile, while others are non-
motile; motile organisms usually have surface appendages
such as cilia, flagella and pseudopodia for movement.
 Some microorganisms have innate ability to synthesizes
their own food from simple organic and inorganic
compounds present in the earth atmosphere. This could be
effected through chemosynthesis or photosynthesis.

3.2 MORPHOLOGICAL CHARACTERISTICS

Bacteria colonies on solid media have distinctive appearances
that are visible to the naked eyes. Such peculiar characteristic
of a colony helps in the preliminary investigation and
identification of a bacterium. These characteristics include the
following
i. Shape of the colony: this could be circular,
irregular, regular, or rhizoidal in shape

ii. Size of the colony: the size varies from tiny colony
to small, to medium and to fairly large and large
colonies depending on the physicochemical
condition of the growth medium and the nutrient
composition.
19
iii. Elevation of the colony: this could be flat, raised,
convex or umbonate depend on the position of the
colony with regards to the growth surface

iv. Edge of the colony: the edge could be entire,

undulate, lobate, detante or rhizoid.

20
 v. Optical characteristics: this could be that the
colony is transparent, translucent or opaque. This is
based on the ability to clearly see through a colony
or faintly see through or that one cannot see through
at all.
vi. Colony surface: this could be smooth, rough, dull,
wrinkled, glistering or granular in nature. This tends
to be a dictate of the colony surface appearance on
the solid media.
vii. Pigmentation of the colony: the description is
based on the colour of the colony on solid media.
The pigmentation (colour) of the colony could be
white, red, pink, light yellow, straw yellow, deep
yellow, orange, cream, milk, dull, green and so on
and so forth.

3.3 BIOCHEMICAL CHARACTERISTICS OF

CLASSIFYING MICROORGANISM
Various biochemical methods used in microbial
identification and their procedures

Biochemical reactions: Whenever there is life, chemical

reactions occur, therefore whenever there is life there is
biochemistry and the versatility of biochemists cannot be over
emphasized. Biochemists, thus study the chemical processes
occurring in microorganisms, plants insects, mammal and
human beings. This concept of biochemistry is also used in
identification of some microorganisms. Example of such
biochemical tests include;
1. Action on simple carbohydrate
2. Starch hydrolysis
3. Action on litmus milk
4. Lipid hydrolysis
5. Protein hydrolysis etc.
21
 CHAPTER FOUR

4.0 GROWTH OF MICROORGANISMS

4.1 FACTORS INFLUENCING MICROBIAL

GROWTH
The growth of microorganisms is greatly influenced by the
chemical and physical nature of the surroundings.
Understanding of environmental effects aids the control of
microbial growth and the study of the ecological distribution of
microorganism. These factors include: Moisture (solute and
water activity), PH, Temperature, Oxygen concentration,
Pressure, Radiation, Nutrients and Growth factors.

Solute and water activity

Osmotic concentration of the environment can effect microbial
growth and survival, if a microorganisms is placed in a
hypotonic solution, water will enter the cell and cause the cell
to burst, but when microorganisms with rigid cell wall are
placed in hypertonic environment water leaves and the plasma
membrane shrink away from the wall, a process known as
plasmolysis occur.

PH (Hydrogen ion Concentration)

PH is a measure of the hydrogen ion concentration of a solution
and is defined as the negative logarithm of the hydrogen ion
concentrate. PH dramatically affects microbial growth. Each
specie has a define PH growth range and pH growth optimum.
Microorganisms can be classified intro three categories based
on their PH tolerance. Acidophiles; are microbes that have their
growth optimum between PH 0 and 5.5. Neutrophiles have
their growth optimum between PH 5.5 and 8.8, and
Alkalophites have their growth optimum between PH 8.5 and
11.5.
22
Temperature
Environmental temperature profoundly affects living things. In
deed microorganisms are particular susceptible to temperature
influence because they are usually unicellular and also
poikiliothermic (their temperature varies with that of external
environment). The most important factor influencing the effect
of temperature on growth is the temperature sensitivity of
enzyme-catalyzed reaction. At low temperature, a rise in
temperature increases the growth rate, because the rate of each
reaction increases, metabolism as a whole is more active at
higher temperature and microorganisms grows fast. But at a
point, high temperature damages microorganisms by
denaturing enzymes, transport carriers and other proteins.
Microbial membranes are also disrupted by heat and the lipid
bilayer simply melts apart.

Microorganisms can be placed in one of five classes based on

their temperature range for growth. These classes include:

(i) Psychrophiles: are those microorganism that grow well at

(00C –150C) the maximum is around 200C examples are genera
of Pseudomonas, Flavobacteria, Achromobacter and
Alkaligenes (ii)Facultative psychrophiles (00C and have
optimum between 20 and 300C and maximum around 350C.
Examples are bacteria and fungi associated with spoilage of
refrigerated foods. (iii) Mesophiles: are microorganisms with
growth optimum temperature around 20 to 450C and a
temperature minimum at 15 to 200C. Their maximum is about
450C or lower. Examples all human pathogen are mesophiles.
(iv)Thermophiles: they can grow at temperature of 550C or
higher. Their growth minimum is usually around 400C and
their optima between 550C and 650C. (v) Hyperthermophiles:
they can grow at 900C or above and some have maxima above
1000C. Bacteria in this class have growth optimum between
23
800C and about 1100C, e.g. Pyrococcus abyssi and
Pyrodictium occultum

Oxygen concentration
Oxygen serves as the terminal electron acceptor for the
electron-transport chain in aerobic respiration. An organism
that can grow in the presence of atmospheric O 2 is call aerobe,
whereas one that can grow in its absence is an anaerobe.

Microorganisms can be grouped into different categories based

on their relationship to O2
1. Obligate aerobes: these are organisms that are completely
dependent on atmospheric O2 for growth
2. Facultative anaerobes: these organisms do not require O2
for growth but do better in the presence of O2
3. Strict or obligate anaerobes: do not tolerate O2 at all and
die in its presences e.g. Clostridium pasteurianun.
4. Aerotolerant anaerobes: these organisms grow well
whether their s oxygen or not e.g. Entorococcus faecalis
5. Microaerophiles: these organisms grow and damaged by
the normal atmospheric level of O2 (20%) and require O2
level below the range of 2 to 10% for growth.

Pressures
Most organisms spend their lives on land or on the surface of
water, where they are always subjected to atmospheric pressure
and yet are never affected significantly by pressure. The
hydrostatic pressure can reach 600 to 1100 atm in deep sea,
while the temperature is about 2 to 30C. Despite these
extremes, bacteria survive and adapt. Based on the adaptation
ability and survival rate of microorganisms under pressure,
microorganisms are categorized as:
a. Barotolerants: are those that can withstand wide range of
atmospheric pressure.
24
b. Barophilic: those that grow more rapidly at high pressure.

Radiation
Our world is bounded with electromagnetic radiation of
various types. Sunlight being the major source of radiation on
the earth, it includes visible light, ultraviolet (UV) radiation,
infrared rays and radio waves. Visible light is most
conspicuous and important aspect of our environment, all lives
are dependent on the ability of photosynthetic organisms to
trap the light energy of the sun.
Nutritional Requirements
Nutrients require by microorganism for metabolic activity are
divided into two namely (i). Macro-element (macro-nutrient)
and (ii) Micro-element (micro nutrients)

Macro-elements; are nutrients required by microorganisms in

relatively large amount. Examples include: carbon, oxygen,
hydrogen, nitrogen, sulfur, phosphorus, potassium calcium,
magnesium and iron

Microelements; are those nutrients required by

microorganisms in trance amount. Examples include:
manganese, zinc, cobalt, molybdenum, nickel and copper.

4.2 MICROBIAL GROWTH

Growth in biological realm can be defined as the orderly and
systematic increase in the quantity of all the chemical
components of the cells.

25
MICROBIAL GROWTH CURVE
Microorganisms are known to exhibit a significant and tangible
growth when introduced into a suitable medium. The reasons
are quite obvious, parts of which include favourable conditions
and desired nutritional materials incorporated into the medium
of growth. However, the inoculation of a bacterium into liquid
culture medium tends to revealed certain growth pattern. This
pattern of growth which is typical of bacteria in a batch culture
is what is referred to as a generalized growth curve of a
bacterium grown in liquid (broth) batch culture tends to show
five different phases of growth. These phases of growth are
represented in the figure bellow.

The graph of microorganisms reproducing by binary fission

can be plotted as the logarithm of cell number versus the
incubation time. The resulting curve has four distinct phases.

C
D
Cell number B
A

Microbial growth curve in a closed system

A = lag phase, B= logarithm phase (exponential phase), C =
stationary phase and D= decline or death phase.
LAG PHASE: in the phase of growth, the growth rate is zero
(0) this is because of the fact that when inoculum is transferred
from an old culture to a new medium, the cells have to readjust
26
to the new environment. Also, it is a vital period during which
the cells increase in size and to synthesis the necessary
enzymes required to metabolize the nutrient provided in the
medium. This phase of growth, marked the period to restore
the composition of ribosomes before protein synthesis can
resume.
LOGARITHM/EXPONENTIAL PHASE: logarithms phase
of growth is also known as exponential growth phase. In this
case, the growth rate is maximum; the cellular division
continues to occur at an exponential rate until it reaches a
constant value. All the cells in this phase of growth are viable,
actively dividing and are of constant size. The cells have
successfully adapted to the newly desired favourable culture
medium.
STATIONARY/STATIC PHASE: this phase of growth is
often referred to as static phase of growth. The growth rate is
zero, simply because the rate at which cells divide is equivalent
to the rate at which cells are dying. This may be brought about
as a result of exhaustion of the nutrients in the medium of
growth, concentration of the end-products of the metabolic
activities, some of which may be toxic or inhibitory or
bactericidal, hence cells are killed. In fact, some may lead to
the lowering of the PH of the medium, making it too acidic for
the cells to tolerate.
DEATH/DECLINE PHASE: Death or the decline phase of
growth is the fourth phase of bacteria growth. The growth rate
is negative. This because more cells are dying than the rate at
which cells are being formed or produced. This could be due to
the accumulation of toxic by-products which must have
increased beyond the tolerable level coupled with the fact that
the nutrients are totally exhausted, hence increase in the death
rate.

27
 CHAPTER FIVE

5.0 ISOLATION, CULTIVATION AND

PRESERVATION OF MICROORGANISMS
5.1 CULTURE MEDIA
Culture medium can be defined as any substrate or materials
that can support the growth of microorganisms. It could be
natural or synthesized one like left over food, soil or garbage
and even remains of plant and animal, culture medium can also
be artificial substrate or specially compounded one.

Culture medium must contain certain essential components or

constituents to be able to support growth of microorganisms.
Importantly, there must be a carbon source usually
carbohydrates, nitrogen source particularly organic one such as
amino acids, peptones and the inorganic like ammonium and
nitrate, some essential growth factors like metallic ions,
vitamins and water which constitute the medium in which
these components dissolve and the form in which they can
easily be taken in by microorganisms for utilization. Hence,
culture medium is any substrate or materials that contain
all the essential nutrients required by microorganisms for
growth. In addition, the formation of the culture medium
should be such that the requirements of the microorganisms are
met with regard to these nutrient components.

Classification on the basis of physical state of media or

consistence
a. liquid media: there are define as aqueous formulation
that do not gel or solidify at temperature above
freezing and that tend to flow freely when the
container inside which they are kept is tilted they are
also known as broths.

28
 b. Semi-solid media: they are not liquid at ordinary
room temperature and exhibit a gelatinous consistence
because they contains an amount of solidifying agent
(gelatin or agar-agar) that harden them slightly but
does not produce a firm substance. Semi-solid media
are used to restrict slightly the movement of motile
microbes. E.g. Cystine Trypticase Agar
c. Solid media: they are media that provide a firm
surface on which cell can form district colony. They
are vital in isolation and sub-culturing bacteria and
fungi. The solid state of these media is due to the
pr4sence of solidifying agent (agar-agar) which is solid
at room and incubation temperature but melts or
liquefy at water boiling temperature (1000C)

Classification on the basis of chemical characteristics

a. Synthetic media (chemically defined): these are media
whose compositions are chemically defined, it contains
highly purified organic or inorganic compound that do
not vary for one source to another and have a molecular
content specified by means of an exact formula.
b. Complex or non-synthetic media: these are media
that contain at least one ingredient that is not
chemically define i.e. a single pure compound and is
not represented by an exact chemical formula and most
of those ingredients are from plant, animals and yeast.
E.g. malt extract from barley malt, meat extract from
beef muscle, yeast extract from baker yeast, blood
serum, milk soybean digest e.t.c.

Culture Media based on the nature of the Nutrients

Based on the nature of the nutrients, culture media can be
classified based on the functions of the nutrients into basic,
enriched (enrichment), selective and differential media.
29
a. Basic Media: These are simple media such as nutrient
agar and nutrient broth that will support the growth of
microorganisms that do not have special nutritional
requirements. They are often used in the preparation of
enriched media, to maintain stock cultures of control
strains of bacteria and for sub culturing microorganisms
from differential or selective media prior to performing
biochemical and serological identification tests.
b. Enrichment media: Is a fluid selective media, which
contains substances that inhibit the growth of unwanted
organisms e.g. Selenite F inhibits Coliform bacilli whilst
allowing the typhoid-paratyphoid organisms to grow
freely, giving enriched culture of these organisms.
c. Selective media: Selective media are media prepared
with substances (e.g. bile salts or other chemicals, dyes,
antibiotics) which inhibit the growth of one organism to
allow the growth of another to be more clearly
demonstrated.
e. Differential (Indicator) media: These are media to which
dyes or other substances are needed to differentiate
microorganism. Many differential media distinguish
between bacteria by incorporating an indicator which
changes colour when acid is produced following
fermentation of a specific carbohydrate e.g MacConkey
agar.

Other examples on the basis of their functions

a. Assay media: these are specially composed media which
are used for the assay of vitamins, amino acid or antibiotic
produced by certain microorganisms
b. Enumeration media: these media that are specifically
employed for detecting microbial content or load of
material such as milk and fruit
30
 c. Maintenance media: these are media formulated to
specifically maintain the viability and physiological
characteristics of microorganisms that may be present in
the sample. These type of media do not contain materials
for microbial optimum, growth.
d. Transport media: these are usually non nutritious media
used for the preservation of organism during their transfer
from one laboratory to another or from one place to
another. Example Ringer solution

5.2 PREPARATION OF CULTURE MEDIA

In the preparation of complex culture media, it is advisable
to use ready-made standard dehydrated media to ensure
good performance and reproducibility. In most cases it will
be less expensive than buying the chemical constituents.
Some chemicals may also be difficult to obtain, not
available in small amount and may have a short shelf life.

Precautionary steps to take while preparing Culture

Media
1. Prepare media made from dehydrated product in a damp
free environment as possible, to prevent the risk of
inhaling fine particle of dehydrated media.
2. Once the ingredients are weighed, do not delay in making
up the medium
3. The container in which the medium is prepared should
have a capacity of at least twice the volume of the
medium being prepared.
4. Use distilled water or deionized water. Water containing
chloride, lead, copper or detergent must not be used.
5. Add the powder ingredients to the water and stir to
dissolve. Do not shake a medium but by stirring or by
rotation the container.

31
 6. When heating is require to dissolve the medium, stir
while heating and control the heat to prevent boiling and
foaming which can be dangerous and damage the
medium. Overheating a medium can alter its nutritional
and getting properties and also its pH. Autoclave a
medium, only when the ingredients are completely
dissolved. Always autoclave at the correct temperature
and for the time specified.

5.3 STERILIZATION OF CULTURE MEDIA

The flowing methods are used to sterilize culture media:
i. Autoclaving, ii. Steaming at 1000C and iii. Filtration

Autoclaving: The majority of culture media are sterilized by

being autoclaved. This ensures the destruction of bacterial
endospores as well as vegetative cells. This should be done at
appropriate temperature, as under autoclaving results in
unsterile medium and over autoclaving causes alteration of the
pH and destruction of essential component of medium.

Steaming: This is used to sterilize media containing ingredient

that would be broken down or inactivated at temperature over
1000C. This can be done in autoclave with the lid left loose or
in form of steam sterilizer. Steaming time vary according to the
type of medium e.g. 15 minute for Cary-Blair medium

Filtration: This provides a means of removing bacteria from

fluids. It is used mainly to sterilize additives that are heat-
sensitive and cannot be autoclaved or less stable substances.
Examples include serum and solutions containing urea and
certain carbohydrates.

32
5.4 TYPES OF CULTURE
There are essentially two types of culture namely (i) Pure
culture and (ii) Mixed culture

PURE CULTURE
This is a culture medium which contains only a single species
of microorganism. Hence, a culture that contains only one
kinds of microorganism at a time is known as pure culture or
axenic culture.

MIXED CULTURE
Materials by nature are exposed to the outside environment
where several species of microorganisms reside. So materials
are known to contain varieties of species of microorganism at a
time. A culture that contains more than one kind of
microorganism is known to be mixed culture. However, if it
contains only two kinds of microorganism deliberately
maintained in association with one another, it is known as two-
member culture. Two member cultures are known to exhibit
syntrophism, in that one of the two members culture tends to
synthesize what the other member lacks needs for growth
particularly essential growth factors.

5.5 ISOLATION OF PURE CULTURE

Pure cultures of microorganisms that form discrete colonies on
solid media may be obtained by modification of the plating
method. This method involves separation and immobilization
of individual organism on or in a nutrient medium solidifying
or jellying agent. After separation, inoculation and incubation
by sub-culturing each viable organism give rise through
growth, to a colony from which subsequent transfer through
aseptic techniques can be readily made.

33
Streaked Plate Method
The streaked plate method appears to be the most widely and
routinely used methods of plating in the laboratory. It involves
the sterilization of wire loop by passing it over blue flame until
it turns reddish. The sterile loop is used to pick a loopful of the
organisms and immediately use to make series of parallel, non-
overlapping streaks on the surface of an already solidified agar
plate. In this event, the inoculum is progressively diluted with
the successive streaks such that even if the initial streaks result
into confluent growth, well isolated discrete colonies
developed along the later streak on the plate. These discrete
colonies on the plate are clearly pure culture and further
subculture can be made from them either for storage and/or for
further microbiological investigation.

Stabbing method: in this method, the solidified agar medium

is cut in such a way that the medium break, this operation is
affected with a sterile wire loop or pin. Stabbing method of
inoculation allows the development of the organisms inside the
medium of growth, thus suitable for the isolation and
cultivation of anaerobic microorganism.

Dropping method: this involves the use of sterilized pipette in

taking known volume of the inoculum culture in liquid
suspension into their molten but cool agar medium or liquid or
broth medium in conical flask or in test tubes plugged with
cotton wool. This method allows the development of the
microbial growth on the surface of the liquid or broth medium
continuously allow not only the even growth of the microbes
but also the equal accessibility of the microbes to the nutrients
contained in the medium of growth.

34
SUBCULTURING
Sub-culturing is the procedure of transferring microbial cells
from their present g-rowth source to a fresh one. When the
transfer is from solid medium (agar) to liquid medium (broth),
the term “picking off” is employed.

Plating out
This is a technique that aims at obtaining single colonies that
are pure for identification from mixed culture.

5.6 MAINTENANCE OF PURE CULTURE

Methods for the maintenance of pure culture in viable
conditions depends on the characteristics of the particular
organisms

Agar slope culture

Slopes may be in test tubes or screw-capped bottle, the latter
being preferable as there is less risk of drying out. Cultures
once grown may be stored in the refrigerator (40C) but must be
subcultured at certain intervals, depending on the organism.

Culture of anaerobic bacteria

These also do not grow on the surface of solid media incubated
aerobically and are best kept in a medium providing reducing
condition e.g. Robertson’s cooked meat medium and
subcultured at interval of up to 1 year.

Freeze died cultures

In this process, the cultures are freeze-dried or lyophilized and
store in sealed glass ampoules under vacuum. Ampoules may
be stores at room temperature or in refrigerator and the culture
will remain viable for prolonged period of several years. This
method useful for storage and dispatch of cultures

35
Preservation under oil
Cultures are first grown in agar slopes and then covered with
sterile liquid paraffin or mineral oil. Culture maintained in this
way will generally remain viable for several years without sub-
culturing

Freezing
This can be achieved directly on the culture medium but for
most organisms, it is necessary to add a pryo-protection agent
such as glycerol/dimethyl suphuroxide before freezing. Better
preservation can be achieved by using liquid Nitrogen
refrigerator maintained at -2000C

Stock Culture
Stock culture is a reserved or kept culture. Pure cultures of
organism obtained are usually kept pure in laboratories for
study and reference and therefore referred to as stock culture.
Stock culture must be maintained so that they are free from
contaminations and retain their viability.

36
 CHAPTER SIX

6.0 CONTROL OF MICROORGANISMS

Microorganisms are generally found everywhere in the
biosphere (where life exist) this includes the soil, air (dust
particles of the atmosphere) , water, surfaces and even the
inner part of animals, plant and other materials.
Microorganisms are known to be responsible for various
diseases in plants and animals. At this juncture, the need to
check various microorganisms and their activities is of
paramount importance. This is being done either by killing or
suppressing/reducing the level of microbial growth on
materials that are of concerned to man.

6.1 REASONS FOR CONTROLLING

MICROORGANISMS

i. To prevent infection of man, his domestic animals and

plant particularly the economic plants.
ii. To stop further spread of infectious and undesired
microorganisms that may be potentially hazardous to man.
iii. To prevent contamination of materials used in pure culture
work in the laboratories such as diagnosis, research and
industries.
iv. To prevent bio-degradation/bio-deterioration and spoilage
of food and other useful items.

6.2 TERMINOLOGIES USED IN CONTROLLING

MICROORGANISMS
 Sterilization: Simply means the destruction of all kinds
and forms of microorganisms

37
  Disinfection: Acts of destroying or inactivating or
removal of microorganism, but this process have no effect
on spore forming organisms, example of such spore
former is Clostridium tetani Disinfectants (e.g. phenol-
based) can be useful in killing many bacteria on certain
instruments, but cannot be used for internal consumption
or on skin. Antiseptics (e.g. iodine or 70% alcohol) are
used topically (e.g. on skin surfaces) to reduce bacterial
load.
 Antisepsis: This is employed when a living tissue is being
disinfected. This is employed to prevent infection.
Example of such is antiseptic. When used for prevention is
called prophylactic and when used for treatment is called
therapeutic.
 Bacteriocidal: Agent that kill bacteria
 Bacteriostatic/inhibitory): Agent that prevents their
growth.
 Sanitizer: Agent that reduces microbial population to a
save level according to public health standard

STERILIZATION
Sterilization refers to complete killing (or removal) of ALL
organisms in a non-selective fashion in including viruses on/in
an object or on/in a material. For example, autoclaving
involves heating liquids (e.g. media) or solids to 121oC under
steam pressure. The materials must be heat resistant.

DISINFECTION: this is the killing or the removal of

organisms that are capable of causing infection. Also involves
the reduction or elimination of pathogenic microorganisms
such that they no longer constitute health hazard. Disinfection
mostly involves uses of chemicals i.e. disinfectants such as
phenol (carbolic acid), formaldehyde, chlorine, iodine or
bichloride of mercury; these are used on inanimate objects or
38
on floors, tiles, dishes, laundry, bedding and work benches,
also used to rinse pipettes, conical flask, test-tubes and bottles
to prevent contamination. In most cases, disinfectants are
poisonous and cannot be used directly on animal tissue or on
human.

6.3 TYPES OF STERILIZATION AND

DISINFECTION

Generally, there are various methods and effective ways of

carrying out sterilization and disinfection. The method use at a
particular time is constrained by the nature of the materials or
objects to be disinfected or sterilized. These includes; (a)
physical disinfection and sterilization and (b) chemical
disinfection and sterilization.

The physical disinfection and sterilization include the

application of heat (dry heat, moist heat and incineration),
irradiation/radiation, filtration, osmotic pressure change,
surface tension variation and electricity application.

Chemical disinfection and sterilization encompasses the

application and the use of chemical substances to achieve these
two cardinal operations.
In some case, a combination of both physical and chemical
methods of disinfection and sterilization may be required to
achieve the ultimate goal of sterility.

39
 REFERENCES

Adam, M.R and Moss, M.O. (1999) Food Microbiology, 3rd

Edition, The Royal Society of Chemistry,
Cambridge
Arotupin, D.J. (1999). Microbiology an introductory approach
1st edition samok printer, Ilorin pp 1-88
Coven, S.T. and Steel, K.J. (1965) Manual for the
Identification of Medical Bacteria, University
Press, Cambridge
Fawole, M.O and Oso, B.A. (1995) Laboratory Manual of
Microbiology, Polygraphic, Press Ltd, Ibadan
Nigeria
Harmful Algal Blooms: Red Tide: Home"(2009)
www.cdc.gov. http://www.cdc.gov/hab/redtide/.
International Human, Genome Sequencing Consortium
(2004). "Finishing the Euchromatic Sequence of the
Human Genome". Nature 431 (7011): 931–945.
Jideani, V.A. and Jineani, I.A. (2006) Laboratory Manual of
Food Bacteriology, Amana Printing and
Advertising Ltd, Kaduna Nigeria.
Prescott, L. M., Harley, J. P. and Klein, D. A. (1996)
Microbiology, third edition, the McGraw-Hill
companies, Inc, Wm. C. Brown Publishers, USA.
Prescott, L.B., Harley, J.P. and Klien, D.A. (1996)
Microbiology, Third Edition, Wm. C. Brown
Publishers, United State of America.
Prescott, L.B., Harley, J.P. and Klien, D.A. (2008)
Microbiology, Seventh Edition, Wm. C. Brown
Publishers, U.S.A.

40