INTRODUCTORY/GENERAL MICROBIOLOGY

(MCB 101/ 108)

BY

FELIX ADE IKUESAN (Ph. D)

What is Microbiology? Microbiology is defined as the study of living organisms that are too
small to be perceived or seen by the unaided or naked human eye but can be viewed or studied
with the aid of microscope. Thus, these small living creatures are said to be microscopic and are
called microscopic organisms, microorganisms or simply microbes. Microorganisms are small
living creatures that cannot be seen with the unaided human eye (they are microscopic in nature).
That is, Microbiology is the study of microorganisms or microbes. Microscopic forms of life are
present in vast numbers in nearly every environment. They are found in the soil, in the bodies of
water, in the food and water we consume and in the air that we breathe and on the surface of our
bodies, in the mouth, nose, and portions of the digestive tract as well as plants. These microbes
include bacteria, rickettsiae, algae, fungi, protozoa and viruses. Microbiology is concerned with
the study of their form, structure, reproduction, identification, physiology and metabolism. It also
deals with the study of their distribution in nature, their relationship with each other and to other
living organisms, their harmful and beneficial effects, the changes which they make in their
environments and the uses to which they can be put. For the most part, microorganisms exhibit
the characteristic features common to biological system; such features as reproduction,
metabolism, growth, irritability etc.
A microbiologist is a specialist in microbiology. Microbiologists study every aspect of
microbes; their genetics, their physiology, characteristics, harmful or beneficial effects, the ways
they interact with the environment and other organisms and their uses in industry, agriculture,
medicine, environment etc.

HISTORY AND SCOPE OF MICROBIOLOGY

Brief history of microbiology
The existence of microbes and their effect was known since ancient times. Lucretius (98-55 BC)
and physician Girolamo Fracastoro (1497-1553) were the first to suggest that invisible
creatures caused disease. Between 1625 and 1630, Francesco Stelluti used a microscope to
study bees and weevils but the start of microbiology as a field of science can be attributed to
Anthonie van Leewenhoek (1632-1723). The brief history of microbiology therefore is as
follows;
 Robert Hooke (1665) - He reported that lives smallest structural units were little boxes
or cells. This marked the beginning of the cell theory; that all living things are composed
of cells.
 Francesco Redi (1668) - Earlier people believed in the “Theory of spontaneous
generation”. He was a strong opponent of the Theory of spontaneous generation. He
demonstrated that maggot appear on decaying meat only when flies are able to lay eggs
on the meat and thus paved the way for disproving the theory of spontaneous generation.
 Anthonie van Leeuwenhoek (1632) - He discovered the invisible world of
microorganisms “animacules”.
 John Needham (1745) - He claimed that microorganisms could arise spontaneously from
heated nutrient broth.
 Lazzaro Spallanzani (1765) - Spallanzani repeated Needham experiment and suggested
that Needham’s results were due to microorganisms in air entering the broth.
 Rudolf Virchow (1858) - He said cells developed from preexisting cells (Biogenesis)
 Louis Pasteur (1822-1895) - His discoveries led to the development of aseptic
techniques used in laboratories and medical procedures to prevent contamination by
microorganisms that are in the air.

Golden age microbiology: Rapid advances in the science of microbiology were made
between 1857 and 1914.

Fermentation and Pasteurization: Louis Pasteur showed that microorganisms (yeasts) are
responsible for fermentation. Fermentation is the conversion of sugar to alcohol to make beer
or wine. Bacterium can oxidize the alcohol and acetic acid.
Pasteur discovered that the growth of microbes cause decomposition of dead plants and
animal tissues. He also showed that microorganisms are responsible for the spoilage of food.
Pasteur also demonstrated that these spoilage microorganisms can be killed by heat that was
not hot enough to evaporate the alcohol in the whine. This application of a high heat for a
short time is called Pasteurization.
 Pasteur contributed to the development of vaccine. He developed vaccines for anthrax and
rabies. He also gave the concept that attenuated bacteria can produce immunity. Attenuated
means weakened virus or bacteria that are unable to cause the disease. Later it was
discovered that killed microbes could also produce immunity.

The Germ theory of disease: Robert Koch provided proof that a bacterium causes anthrax
and provided the experimental steps known as Koch’s postulates to prove that a specific
microbe causes a specific disease.

Koch’s postulates:
 Pathogens must be present in all cases of disease
 Pathogens must be isolated and grown in laboratory in all pure culture
 Pathogens from pure culture must cause the same disease when inoculated into healthy
susceptible laboratory animal
 Same pathogen must be isolated from a diseased animal

Vaccination:
Immunity is conferred by inoculation with a vaccine. Edward Jenner (1978) is credited with the
discovery of vaccine. He demonstrated that inoculation with cowpox material provides human with
immunity from small pox. Pasteur discovered that avirulent bacteria could be used as vaccine
for chicken cholera, he coined the word vaccine. The term vaccination thus came from vacca
for cow and was coined by Pasteur in honor of Edward Jenner.

Modern chemotherapy
Treatment with chemicals is called chemotherapy. Paul Ehrlich (1908) gave the principle of
chemotherapy or selective toxicity (the drug must be toxic to the infecting microbe, but
relatively harmless to the host cell). Paul Ehrlich developed a synthetic arsenic drug,
salvarsan to treat syphilis. Chemotherapeutic agents used to treat infectious disease can be
synthetic drugs or antibiotics. Antibiotics are chemical produced by bacteria and fungi that
inhibit or kill other microbes.
 Alexander Fleming (1928): He discovered the first antibiotic from Penicillium notatum
(fungus) that killed staphylococcus aureus isolated from wounds. Penicillin was tested
clinically and mass produced.
The Discovery of microorganism or The microbial world.
The discovery of invisible creatures was not generally accepted until 1677 when Anthonie van
Leeuwenhoek (believed to be the first person to have made a powerful glass lenses) observed and
described the bacteria. He called them “animalcules”. The start of microbiology as a field of science can
be attributed to Anthonie van Leeuwenhoek (1632-1723).
Leeuwenhoek (born 24th October 1632) is therefore known as the Father of Microbiology. He was a
cloth merchant in Delft Holland. He had little or no formal education but was armed with lot of patience
and curiosity with glass cutting as his hobby, which he financed from his business. He made hand lenses
powerful enough to examine microorganisms from different habitats. His unending curiosity led to him to
spend hours examining specimens collected from lake, rain waters, saliva, infusion, both scratching even
from his own teeth and those of others. He made descriptions of his observations which were accurate
enough and included all forms of bacteria known today.

The magnifying power of his early instrument ranged from approximately 50 to 300 times the diameter of
a particular specimen. Leeuwenhoek’s contribution to the development of microbiology has been
formally established because of his remarkable observations and description of microscopic forms of life.
Among his first observation were description of protozoa and of basic shapes of bacteria, yeast and algae.

Leeuwenhoek recognized the value of recording his observations. He sent over 200 handwritten,
occasionally illustrated letters to the Royal Society of London and published in English.

With more improvement and development, microscope became generally available in the 19 th century
when discoveries of Leeuwenhoek were confirmed by Robert Hooke who later developed the compound
microscope.
Scope of microbiology.
Generally speaking, microbiology is a wide subject and has influence on genetics, agriculture,
food science, ecology, immunology, medicine and various fields but can be grouped into;
(i) Pure microbiology e.g. Bacteriology, Mycology, Phycology, Virology, Protozology,
Immunology, Microbial cytology, Microbial physiology, Microbial genetics,
 Microbial ecology, Microbial taxonomy, Cellular Microbiology and Molecular
Microbiology
(ii) Applied microbiology e.g. Medical Microbiology, Veterinary Microbiology, Public
Health Microbiology, Industrial Microbiology, Pharmaceutical Microbiology,
Agricultural Microbiology (Plant Microbiology & Soil Microbiology), Food & Dairy
Microbiology, Environmental Microbiology, Microbial Biotechnology
Roles of microorganisms
Mankind and nature generally is affected by the activities of these invisible creatures. Among the
importance of microorganisms to mankind include the following:
The environment:
 Microorganisms are responsible for the cycling of carbon, nitrogen, phosphorus
(geochemical cycles). Microorganisms are involved in the destruction of materials
especially in humid environment. Such materials as clothing, leather shoes, woods and
wooding materials e.t.c. are easily damaged by microorganisms.
 Maintain ecological balance on earth.
 They are found in association with plants in symbiotic relationships, maintain soil
fertility and may also be used to clean up the environment of toxic compounds
(bioremediation)
 Microorganisms are useful in biodegradation. They help to degrade various materials
such as leave drops from trees, corps of animals which otherwise would have littered our
environment.
 Some are devasting plant pathogens, but others act as biological control agents against
these diseases.
Roles of Microbes in medicine and Public Health
 It has been known since long time that microorganisms in the human body play an
important role in maintaining human health. Certain microbes can help us fight against
other microbes.
 A few harmful microbes, for example less than 1% of bacteria, can invade our body (the
host) and make us ill. Microbes cause infectious disease (they are pathogenic) e.g. small
pox caused by Variola virus, Cholera caused by Vibrio cholerae, Syphilis caused by
Treponema pallidium, Tetanus by Clostridium tetani, HIV/ AIDS by Human
 Immunodeficiency Virus, Leprosy and Tuberculosis by Mycobacterium leprae and
Mycobacterium tuberculosis respectively. Malaria caused by Plasmodium. There is also
strong evidence that microbes may contribute to non- infectious chronic diseases such as
some forms of cancer and coronary heart disease. Different diseases are caused by
different types of microorganisms. Microbes that cause disease are called pathogens.
 Microbes are also responsible for food borne diseases
 Microorganisms are also used in the production of vaccines e.g BCG used in
immunization against tuberculosis.
 Microorganisms are of medical importance as therapeutic agents in the treatment of
infectious diseases e.g. the production of antibiotics.
 Microorganisms also cause food spoilage.

Infectious Microbe that causes the Type of

disease disease microbe
Cold Rhinovirus Virus
Chickenpox Varicella zoster Virus
German measles Rubella Virus
Whooping cough Bordatella pertusis Bacterium
Bubonic plague Yersina pestis Bacterium
TB Mycobacterium tuberculosis Bacterium
(Tuberculosis)
Malaria Plasmodium falciparum Protozoan
Ring worm Trichophyton rubrum Fungus

Athletes foot Trichophyton mentagrophyte Fungus

It is important to remember that:

 A pathogen is a microorganism that has the potential to cause disease.
 An infection is the invasion and multiplication of pathogenic microbes in an individual or
population.
 Disease is when the infection causes damage to the individual’s vital functions or
systems.
  An infection does not always result in disease.
To cause an infection, microbes must enter the bodies. The site at which they enter is known as
portal of entry. Microbes can enter the body through any of the sites listed below:
 Respiratory tracts (mouth and nose) e.g influenza virus which causes flu
 Gastrointestinal tracts (mouth oral cavity) e.g. Vibrio chlorae which causes cholera.
 Urogenital tract e.g. Escherichia coli which causes cystitis.
 Eyes
 Breaks in the skin surface e.g Clostridium tetani which causes tetanus.
To make people ill, microbes have to:
 Reach their target site in the body;
 Attach to the target site they are trying to infect so that they are not dislodged;
 Multiply rapidly;
 Obtain their nutrient from their host;
 Avoid and survive attack by the host’s immune system.

Food/Industry:
 Microorganisms have been used to produce food, from brewing and wine making,
through cheese production and bread making, to manufacture of soy sauce and single cell
protein.
 Microbes are also responsible for food spoilage.
 In the pharmaceutical industry and medicine, they are employed for the production of
several drugs and enzymes used in the treatment of different human and animal ailments
e.g antibiotics, steroids, alcohols, vitamins and amino acids etc.
Biotechnology:
 Commercial applications include the synthesis of acetone, organic acids, enzymes,
alcohol and many drugs.
 Genetic engineering- bacteria can produce important therapeutic substances such as
insulin, human growth hormone and interferon.
Research:
 Because of the simple structure of microorganisms, they are easier to study most life
processes in simple unicellular organisms than in complex ones
  Millions of copies of the same single cell can be produced in large number very quickly
and at low cost to give plenty of homogenous experimental materials.
 Because they produce very quickly, they are useful for studies involving the transfer of
genetic information
Contribution of some Microbiologists in the development of public health
(a) Louis Pasteur (1822- 1895):
(i) Proving the germs’ theory of disease was the crowning achievement of Louis
Pasteur. That is, he demonstrated that microorganisms cause diseases
(ii) He discovered how to make vaccines from weakened, or attenuated microbes eg
vaccines against rabies
(iii) He found that environment directly affected contagion
(iv) Spread of diseases can be controlled by sterilization
(b) Robert Koch (1843- 1910):
(i) He was one of the founders of bacteriology
(ii) He discovered the anthrax disease cycle
(iii) He discovered the bacteria responsible for tuberculosis and cholera
(iv) He was the proponent of the Koch’s postulate. Koch’s postulate is a guideline to
prove that a specific disease is caused by specific organism
(v) Pure culture techniques were perfected by Koch.
(c) Antonie van Leeuwen1hoek (1632- 1723):
(i) He is considered to be the father of Microbiology
(ii) He was one of the first people to observe microorganisms using a microscope of his
own design. The discovered the existence of microorganisms
(iii) He was the first to observe bacteria and protozoa
(d) Sir Alexander Flemming (1881- 1955)
(i) He was the first to discover the first broadly effective antibiotic substance which he
named penicillin
(ii) He discovered the enzyme lysosome.
(e) Dr John Snow (1813-1858)
(i) A leader in the development of anesthesia and medical hygiene
(ii) Established cholera as a water – borne disease
 (iii) Pioneer of epidemiology. He is the father of modern epidemiology.
(f) Edward Jenner (1749-1823)
(i) Pioneered the concept of vaccine
(ii) Known for smallpox vaccine
(iii) Discoverer of vaccination for smallpox

MICROSCOPE AND MICROSCOPY

Microbiology and Microscopic techniques
Microscope: The word microscope was coined by Giovanni in 1625. Microscope is an optical
instrument comprised of one or more lenses and it is used to enlarge and or magnify images of
microscopic objects.
Microscopy: This the technical field of using microscope to view samples and objects that
cannot be seen with the unaided eyes (objects that are not within the resolution range of the
normal eye).
Types of microscopy
 Light or optical microscopy: Uses visible or UV light to illuminate the objective. Light
passes through a series of lenses, which alter the path of light and produced a magnified
image of the object
 Electron microscopy: Uses a beam of electrons to illuminate the objectives. The
electron beam is moved around by magnets, which act like lenses in an ordinary
microscope. Electron microscope can magnify objects to over 200,000 times.
 Scanning probe microscopy
 X-ray microscopy
Principles of microscopy
Image magnification and magnifying power: The main purpose of microscopy is image
magnification. Image magnification depends on;
 Magnifying power of the objective and eyepiece
 Numerical aperture and the resolution power of the instrument
 Wavelength of light
In a light microscope, a series of ground lenses bend the visible light to magnify the image of the
object. The use of two convex- convex lenses i.e ocular and objective enlarge the image. The
objective magnifying power is 4X to 100X and ocular or eye piece magnifying power is 10X to
20X. Therefore, total magnification is equal to the product of the magnifying power of both
lenses.

Power of objective X Power of ocular Total magnifying power

10X low power 20X 200X
40X high dry power 10X 400X
100X oil immersion 10X 1000X

The 10X, 20X and 40X objectives are dry, meaning that there is air between lens and the slide.
The 100X objective is also called immersion objective as it is used by immersing objective lens
in oil. The advantage is that the working distance between the object and objective is reduced
and the refractive index of the medium i.e oil is better than air and water, resolving power (RP) is
improved and the image is highly magnified, therefore 100X is used to observe bacteria
The Light Microscope
This is one of the basic equipment used by Microbiologists. The simplest microscope operates on
the principle of the magnifying glass, however most modern microscopes are compound
microscopes. The compound microscope is one whose initial image, (i.e. the magnified image
formed by the objective lens), is further enlarged by another lens (the ocular), which produces a
highly enlarged virtual image of the object. When the magnified virtual image produced by the
objective lens is viewed through one eyepiece, the microscope is called a monocular
microscope but when it has two eye pieces, it is a binocular microscope
 Monocular Microscope
The light microscope could be illuminated by daylight reflected into a mirror located below the
sub stage condenser. However, most modern light microscopes use various lighting systems
some of which are highly sophisticated. Some of the more frequently used systems include:
fluorescence microscopes etc.
Classification of Microscope
(1) Based on the number of lenses: microscope can be classified into two types; (i) simple
microscope (ii) compound microscope
(2) Based on the principle involved in magnification, there are two types; (i) Light
microscope e.g bright field, dark field, fluorescence, phase contrast etc (ii) Electron
microscope: use a beam of electrons instead of light for magnification. There are three
types of electron microscope: Transmission Electron Microscope (TEM), Scanning
Electron Microscope (SEM) and Scanning Tunneling Microscope.
Components of the light microscope
The light microscope consists of mechanical and optical components.
1. Mechanical components: These components are required for operating the instrument
and include
i. Stand and frame for all parts
ii. Base or foot is horse-shoes shaped and support the stand
iii. Stage: the microscope stage is a flat plate on which the specimen (usually
mounted onto a glass slide) is placed for observation. It is attached to the top of
 the pillar. It has a circular opening in the center called aperture. It is also equipped
with mechanical devices that holds the specimen slide in place and can smoothly
move the slide back and forth as well as from side to side.
iv. Sub stage is positioned below the stage and holds the condenser.
v. Body tube. This is hollow, cylindrical, usually having a standard diameter. It is
attached to the arm in such a manner that it’s in line with the stage aperture. It
carries the ocular on the top. It can be moved up and down by the screws or
focusing knobs. The mechanical tube length of an optical microscope is defined
as the distance from the nosepiece opening where the objective is mounted to the
top edge of observation tube where where the eyepiece (ocular) are inserted.
vi. Focusing knobs are attached to the body tube. They help focus the image of the
specimen through the eyepiece and are of two types:
(a) Coarse adjustment knob with a micrometer head.
(b) Fine adjustment knob with a micrometer screw head. It is used to sharpen the
image.
vii. Nosepiece is a saucer-shaped structure and it is attached to the base of the body
tube. It has fixed place where the objective can be attached. The whole assembly
i.e nosepiece with the objective can revolve in such a manner that the objective
can be changed according to the required magnification.
2. Optical components: These are required for image formation and include:
i. Mirror: is mounted on a frame and attached to the pillar just below the condenser
It has two surfaces, one plane and the other concave. It can rotate freely and can
be focused in three different directions. It collects and direct the beam of light
used to illuminate the specimen. Modern microscopes have a fived illumination
system.
ii. Objectives; determines the quality of images that the microscope is capable of
producing. Usually, they are of three magnifications 10x, 40x, and 100x (oil
immersion), but objectives of other magnifications can also be used. Objective
lens is usually a compound lens made from different kind of glass. Water
immersion objectives uses water in the place of oil as the immersion medium.
Objectives are classified into the following classes based on degree of correction.
 a. Non chromatic: without correction
b. Achromatic: with improved correction. These compound objectives are used
for routine microscopy. They correct both distortion and aberration.
c. Apochromatic: with greatly improved correction. It reduces chromatic
aberration as well as spherical aberration. Chromatic aberration is more finely
corrected which produces high quality image revealing the true color of the
specimen without distorting the image (used mainly for photography).
iii. Ocular lens or eyepiece in combination with objectives further magnifies the
image. Eyepiece are usually of standard dimension and are made up of two or
more lenses; upper component or eye lens is the magnifier while the lower
component is called field lens. Lenses of variable power can be exchanged with
each other. Eyepieces are also designed to work together with objectives to
eliminate chromatic aberrations. Eyepieces are available in different
magnifications e.g. 1x, 2x, 5x, 10x, 15x, 20x.
iv. Objective condenser collars allow adjustment for fluctuation in cover glass
thickness.
v. Condenser fills the objective evenly with a cone of light. The cone of light is
narrow for the objective of low NA ( e.g. 0.25) but needs to be very wide for one
of high NA ( e.g. 1.25). The cone of light exiting from the condenser must be
adjusted with the condenser iris diaphragm. Condensers are of the following
types:
a. Abbe-type: cheap but have a poor image formation quality.
b. Aplanatic: less expensive yield good illumination.
c. Achromatic-aplanatic: expensive but gives best illumination
d. Dark-field condensers: illuminates the object “from the side” instead of
“straight through” as in bright field.
e. Phase contrast condensers: are actually of the simplest uncorrected type and
only differ from the ordinary condenser in having a set of ring- shaped
“annular stops” one for each phase- contrast objective, centerable and
mounted in a rotating disc.
 f. Interphase- contrast condenser: are of complex optical design and are used in
conjunction with additional special components in the microscope.
 Filter holder: Holds the color filters used to change the wavelength of
light
 Iris diaphragm is attached to the condenser and regulates the light
reflected from the mirror into the condenser and on to the object.

Distortion and optical aberration.

When only one lens is used, we often get image distortion. Distortion of image is known as
optical aberration. When we use a compound lens, the second lens corrects any aberration from
the first lens.
Optical aberrations are of two types:
1. Spherical aberration: distortion based on the shape lens.
2. Chromatic aberration: distortion based on the color of light, as the amount of all the
colors making up light are not refracted the same amount when passing through a glass
lens.

Correction of optical aberration.

Distortion can be corrected by using compound lenses i.e. lens of different shapes and
compositions.
Assignment
a. Draw a well labeled Binocular microscope.
b. State the functions of the various parts labeled.
How to use a compound microscope
Procedure and observation
1 Plug the microscope to the main supply and switch on the light.
2 Place a slide with mounted specimen on the stage of the microscope with the side containing
the specimen facing upwards.
3 Adjust the slide such that the area to be viewed is directly over the hole in the center of the
stage.
4 Locate specimen using the low power objective.
5 Under the X40 objective and while watching the objective lens and the stage from the side.
6 Lower the body tube or raise the stage by means of the coarse adjustment until the objective
is very close to, but not touching the side.
7 While looking through the eyepiece, slowly raise the objective or lower the stage by means
of the coarse adjustment until you can see the specimen well.
8 Then focus with the fine adjustment to obtain a sharp image.
9 To obtain optimum light intensity adjust the condenser and the iris diaphragm.
10 Examine the specimen and make drawings as appropriate.
How to use the oil – immersion objective
Procedure and Observation
1. Follow steps 1 to 7 under the procedure above
2. Adjust the slide to position the specimen to be examined exactly at the center of the X40
objective field.
3. Raise the body tube or lower the stage and slowly rotate the nosepiece until the oil-
immersion objective clicks into position.
4. Put a drop of immersion oil on the portion of the slide directly under the objective.
5. Carefully lower the objective until it touches the oil, but not the slide, while you are
watching from the side.
6. Observe through the eyepiece and bring specimen into focus with the fine adjustment.
7. Make drawing as appropriate.
Precautions in the use of a Microscope
The following points should be noted when handling and using a microscope.
(a) Never lower the body tube or raise the stage with the coarse adjustment while you are looking
through the eyepiece so that objective may not break the coverslip or slide

(b) Never introduce any liquid onto stage; always wipe the lower side of your slide dry before
placing it on the stage
(c) Never use the X40 objective without a coverslip on the specimen.
(d) Always remove all slide from the microscope when not use.
(e) Do not tilt the microscope when working with oil- immersion objective to avoid the oil
flowing under the mechanical stage and onto the sub stage condenser.
(f) Use proper immersion liquid on immersion objectives.
(g) Never use immersion liquid on non-immersion objectives as it can damage the lens
mounting glue After using the oil-immersion objective always clean the oil from the
objective immediately with lens tissue.
(h) On moving the microscope from one place to another, always carry it by the limb with one
hand while supporting the foot with other.
(i) After use, always store your microscope with the X10 objective in the focusing position.
(j) Always protect the microscope from dust by covering with the provided cover.
(k) Never blow on the lens as it deposits saliva on it.
(l) Never clean the lens with a dry lens cleaning paper but use lens tissue
(m) Never use facial tissue paper or bibulous paper to clean lenses as they may contain glass
filaments, which can stretch lenses.
(n) Stubborn stains can be removed by xylene but lens mounty glues are soluble in alcohol,
therefore use xylene sparingly
(o) Do not touch lenses since finger print degrade image quality
(p) Internal optics should always be professionally serviced when needed.
(q) Avoid extreme temperature and high humidity in work areas.
(r) Store the microscope in circulating air.
(s) In very high humidity, store optical parts in the covered containers with desiccant and keep
them very clean to prevent mold growth on the optic coating.
(t) Never use sub stage diaphragm to control brightness.
(u) Wipe body of the microscope with a soft cloth dipped in alcohol or soap water.
(v) Keep sliding parts lubricated with a petroleum jelly or manufacturers recommended
lubricant.

GENERAL CELL STRUCTURE

Types of Cell: Living organisms are made up of cells, Robert Hooke was the first person to
describe cells on the basis of his study of a cork slices. His discovery led to the formation of cell
theory by Theodore Schwann and Mathias Schiedam (1838-1939).
The cell theory stated that;
1. All living organisms are made up of cells.
2. Cell is the basic unit of structure for all living organisms.
 3. All cells arise from preexisting cells.

There are mainly two types of cells (viruses, prions, viroids are acellular; without a cell).
Because of their characteristics, microorganisms join all other living organisms in two major groups
of organisms: prokaryotes and eukaryotes . Microorganisms consist basically of prokaryotes and
microscopic eukaryotes. Bacteria are prokaryotes (simple organisms having no nucleus or
organelles) because of their cellular properties, while other microorganisms such as fungi, protozoa,
and unicellular algae are eukaryotes (more complex organisms whose cells have a nucleus and
organelles). Viruses are neither prokaryotes nor eukaryotes because of their simplicity and unique
characteristics. Microorganisms with the prokaryotic cell structure are devoid of nuclear
membrane and are referred to as lower protists and include Bacteria or Schizomycetes,
Mycoplasma, Rickettsiaae, Chlamydiae, Algae (some the blue green algae called Cynophyta or
Cyanophyceae). That is, prokaryotic cells are cells in which internal membrane bound structures
are absent (i.e. no membrane - bound nucleus or membrane - bound organelles. Organisms of the
kingdom Monera are prokaryotic. All the organisms are unicellular and microscopic and
resemble each other closely with minor difference hence; usually bacteria are taken as a
representative member of this group of organisms.
 Structur
e of

prokaryotic cell

Structure of Eukaryotic cell

Eukaryotic cells (True nucleus): Cells in which internal membrane – bound structures
(membrane - bound nucleus or membrane - bound organelles) are present. These organisms with
the Eukaryotes cell structure are referred to as higher protists and consists of the following
organisms, Algae (moss), Protozoa, Slime mold, Fungi proper or Eumycetes including yeasts and
molds and animals.
The five kingdoms.
The generally accepted classification of living things was devised by Robert Whittaker of Cornell
University in 1969. Whittaker suggested a five-kingdom classification.
The five kingdoms are
 Monera (in some books, Prokaryotae). Prokaryotes, such as bacteria and cyanobacteria
(formerly, blue-green algae), are in this kingdom; the second kingdom,
 Protista: This includes protozoa, unicellular algae, and slime molds, all of which are
eukaryotes and single-celled;
 Fungi: these are the molds, mushrooms, and yeasts. These organisms are eukaryotes that
absorb simple nutrients from the soil
 Plantae (plants) and
 Animalia (animals).
Prokaryotes and eukaryotes are distinguished on the basis of their cellular characteristics. For
example, prokaryotic cells lack a nucleus and other membrane-bound structures known as
organelles, while eukaryotic cells have both a nucleus and organelles. The important cellular features
of a prokaryotic cell (a bacterium) and eukaryotic cell are summarized below.
Differences between prokaryotic Cells and Eukaryotic Cells

Properties Prokaryotic Eukaryotic

1 Endoplasmic reticulum Absent Present

2 Golgi body Absent Present

3 Mitochondria Absent Present

4 Ribosomes Present, 70s 2types present: 70s and

80s

5 Nucleo plasma Absent Present

6 Cell membrane contain Absent Present

steroids

7 DNA presence in organelle Absent Present

8 Chloroplast Absent Present and absent

9 Histones Absent Present

10 Nucleolus Absent Present

11 Membrane Absent Present

12 Chromosomes 1 circular > 1, typically rod during

chromosome mitosis

13 Nuclear division Not by mitosis By mitosis

14 Peptidoglycan in cell wall Present Absent

15 Plastids Absent Present

Prokaryotic and eukaryotic cells are similar in several ways. Both types of cells are enclosed by
cell membranes (plasma membranes), and both use DNA for their genetic information. Viruses are
considered neither prokaryotes nor eukaryotes because they lack the characteristics of living things,
except the ability to replicate (which they accomplish only in living cells).
Classes and characteristics of microorganisms
Microorganisms are divided into seven types viz;
  bacteria,
 archaea,
 protozoa,
 algae,
 fungi,
 viruses and
 multicellular animal parasites (helminths).
Each type has a characteristics cellular composition, morphology, means of locomotion and
reproduction
Bacteria: Bacteria are microscopic and unicellular organisms. The cells are described as
prokaryotic because they lack a nucleus. They exist in four major shapes: bacillus (rod shape),
coccus (spherical shape), spirilla (spiral shape) and vibrio (curved shape). Most bacteria have a
peptidoglycan cell wall, they divide by binary fission and they possess flagella for motility
Flagellum structure
(a) Monotrichous Flagellum

(b) Amphitrichous flagellum

Amphitrichous flagella are located at each end of the bacterial cell.

(c) Lophotrichous Flagellum

The flagella in a lophotrichous arrangement are situated on one end of the bacterial cell, as with a
monotrichous flagellum.
 (d) Peritrichous Flagellum

The peritrichous flagella are located randomly over the surface of the bacterial cell. Many
flagella exist in the peritrichous orientation, and can cover the entire cell.

The difference in their cell wall structure is a major feature used in classifying these organisms.
According to their cell wall structure stains bacteria can be classified as either Gram positive or
Gran negative when using the Gram staining. Bacteria can be further divided based on their
response to gaseous oxygen into the following groups: aerobic (living in the presence of
oxygen), anaerobic (living without oxygen) and facultative anaerobes (can live with or without
oxygen).
According to the way they obtain energy, bacteria are classified as heterotrophy or autotroph.
Autotrophs make their own food using energy of sunlight or chemical reaction in which they are
called chemoautotrophs. Heterotrophs obtain their energy by consuming other organisms.
Bacteria that use decaying life forms as source of energy are called saprophytes.
Archaea
Archaea or Archaebacteria are different from true bacteria in their cell wall structure and lack
peptidoglycans. They are prokaryotic cells with avidity to extreme environmental conditions.
Based on their habitat, all Archaeans can be divided into the following groups:
 Methanogens (methane- producing organisms)
 Halophiles (archaean that live in salty environments)
 Thermophiles (archaean that live at extremely hot temperature)
 Psychrophiles (cold temperature Archaeans)
Archaeans use different energy sources like hydrogen gas, carbon dioxide and sulphur. Some of
them use sunlight to make energy, but not the same way as plants do. They absorb sunlight using
their membrane pigment bacteriorhodopsin. This reacts with light, leading to the formation of
energy molecule adenosine triphosphate (ATP).
Fungi
Fungi (mushroom, mold and yeasts) are eukaryotic cells (with a true nucleus). Most fungi are
multicellular and their cell wall is composed of chitin. They obtain nutrients by absorbing
organic material from the environment (decomposers), through symbiotic relationship with
plants (symbionts), or harmful relationship with a host (parasites). They form characteristic
filamentous tubes called hyphae that help absorb nutrients. The collection of hyphae is called
mycelium. Fungi reproduce by releasing spores.
Protozoa
Protozoa are unicellular aerobic eukaryotes. They have a nucleus, complex organelles and obtain
nutrients by absorption or ingestion through specializes structures. Protozoa have been
traditionally divided based on their mode of locomotion: flagellates produce their own food and
use their whip- like structure to propel forward, Ciliates have tiny hair that beat to produce
movement. Amoeboids have false feet or pseudopodia used for feeding and locomotion and
sporozoa are non- motile. They also have different means of nutrition which groups them as
autotrophs or heterotrophs.
Algae
Algae, also called cynobacteria or blue- green algae are unicellular or multicellular eukaryotes
that obtain nourishment by photosynthesis. They live in water, damp soil and rocks and produce
oxygen and carbohydrates used by other organisms. It s believed that cyanobacteria are the
origin of green land plants.
Viruses
Viruses are a unique group of infectious agents whose distinctiveness resides in their simple,
acellular organization and pattern of reproduction. A complete virus particle or virion consists of
one or more molecules of DNA or RNA enclosed in a coat of protein, and sometimes also in
other layers. These additional layers may be very complex and contain carbohydrates, lipids, and
additional proteins. Viruses can exist in two phases: extracellular and intracellular. In the
extracellular phase called virions, they possess few if any enzymes and cannot reproduce
independent of living cells. While in the intracellular phase, viruses exist primarily as replicating
nucleic acids that induce host metabolism to synthesize virion components; eventually complete
virus particles or virions are released. The virus particle, called a virions or nucleocapsid, is not a
cell. In summary, viruses differ from living cells in at least three ways: (i) their simple, acellular
organization; (ii) the presence of either DNA or RNA, but not both, in almost all virions (human
cytomegalovirus has a DNA genome and four mRNAs); and (iii) their inability to reproduce
independent of cells and carry out cell division as prokaryotes and eukaryotes do.
Generally, viruses have the following properties:
1. They are small (submicroscopic) and can only be viewed with the electron microscope.

2. They have a simple design or architecture, which consists of a nucleic acid core and outer
protein coat.

3. They contain only kind of nucleic acid either DNA or RNA, which serve as the genome. The
nucleic acid can be one of any four forms: dsDNA, ssRNA, ssDNA or dsRNA.

4. They are the only organisms in which the RNA serves as hereditary material.

5. They are obligate intracellular parasites and lack metabolic mechanism and enzyme system.
Therefore, they are resistant to antibiotics, which act against metabolic pathway.

6. They can form crystal unlike other organisms and impure chemicals.

7. They are incapable of self-reproduction but take advantage of a living host cell to replicate.
Their components are produced separately and then assembled into a mature virion.

8. They show a high level of host specificity, e.g. animal virus cannot infect plants.

9. Some viruses can cause diseases in animals and plants.

GROWTH OF MICROORGANISMS
The growth of microorganisms is a highly complex and coordinated process, ultimately
expressed by increase in cell number or cell mass. Growth is defined as an increase in the
number of cells in a population of microorganisms. It is an increase in cellular constituents
leading to a rise in cell number when microorganisms reproduce by processes like binary fission
or budding.
A microbial cell has a lifespan and a species is maintained only as a result of continued growth in
its population. Growth is the ultimate process in the life of a cell- one cell becoming two and
subsequently leading to an increase in the number in a population of microorganisms. In general,
when all other conditions are kept ideal, growth of the microorganisms is dependent on the
substrate (nutrient) supply and their transport into the cells and the environmental factors such as
aeration, O2 supply, temperature and pH. In Microbiology, growth is synonymous to
reproduction. Most prokaryotes reproduce by binary fission, budding or fragmentation.

PREPARATION OF CULTURE MEDIA FOR MICROBIAL GROWTH

A culture medium is any material prepared for the growth of bacteria in a laboratory.
Microbes that grow and multiply in or on a culture medium are known as a culture. Agar is a
common solidifying agent for a culture medium.

Microbiological culture media could be classified according to:

1. Consistency, which could be adjusted by changing the concentration of solidifying or

gelling agents, e.g. agar, gelatin (liquid media do not contain such materials)
 Cultures in liquid media (or broth) are usually handled in tubes or flasks and
incubated under static or shaken conditions. This way, homogenous conditions are
generated for growth and metabolism studies,(e.g. with the control of optical
density and allowing sampling for the analysis of metabolic products).
 Semisolid media are usually used in fermentation and cell mobility studies, and
are also suitable for promoting anaerobic growth.
 Solid media are prepared in test tubes or in Petri dishes, in the latter case, the solid
medium is called agar plate. In the case of tubes, medium is solidified in a slanted
position, which is called agar slant, or in an upright position, which is called agar
deep tube. Solid media are used to determine colony morphology, isolate cultures,
enumerate and isolate bacteria (e.g. using dilutions from a mixed bacterial
population in combination with spreading), and for the detection of specific
biochemical reactions (e.g. metabolic activities connected with diffusing
extracellular enzymes that act with insoluble substrates of the agar medium).
2. Composition
  Chemically-defined (or synthetic) media are composed only of pure chemicals
with defined quantity and quality.
 Complex (or non-synthetic) media are composed of complex materials, e.g. yeast
extract, beef extract and peptone (partially digested protein), therefore their
chemical composition is poorly defined. On the other hand, these materials are
rich in nutrients and vitamins.
3. Function
 All-purpose media do not contain any special additives and they aim to support
the growth of most bacteria.
 Selective media enhance the growth of certain organisms while inhibit others due
to the inclusion of particular substrate(s).
 Differential media allow identification of microorganisms usually through their
unique (and visible) physiological reactions. In the detection of common
pathogens, most practical media are both selective and differential.
 Enrichment media contain specific growth factors that allow the growth of
metabolically fastidious microorganisms. An enrichment culture is obtained with
selected media and incubation conditions to isolate the microorganisms of
interest.

CULTURING OF MICROBES
Culture: A culture is the microorganisms that grow in a culture medium. To culture means,to
grow microorganisms in culture medium.
Culture medium [pl. media]: Culture media are solutions containing all of the nutrients and
necessary physical growth parameters necessary for microbial growth. Note that not all
microorganisms can grow in any given culture medium and, in fact, many can't grow in any
known culture medium. In addition to chemical and physical characteristics, media can be
distinguished qualitatively as:
i. solid and broth
ii. non-synthetic and chemically defined
iii. reducing
iv. selective
 v. differential
Solid medium [agar]: Solid media contain agar (mostly inert solidifying agent). Solid medium has
physical structure (broth lacks structure) and this allows bacteria to grow in physically informative
or useful ways (e.g., as colonies or in streaks). Solid medium is usually used as:
 slants
 stabs
 petri dishes

Colony: A colony is a pile or mass of a sufficiently large number of cells, growing on or in solid
medium, that they are visible to the naked eye.
Enriched medium therefore may be chemically defined or chemically undefined, simple or
complex.
An example of enriched medium employing the latter would be blood agar which is made from
complex medium to which whole blood has been added.
Selective medium
Selective medium is designed to suppress the growth of some microorganisms while allowing
the growth of others (i.e., they select for certain microbes).
Solid medium is employed with selective medium so that individual colonies may be isolated.
Examples of selective media include:
mannitol salts agar (selects against non-skin flora)
 MacConkey agar (selects against gram-positives)
eosin-methylene blue agar (selects against gram-positives)
phenylehyl alcohol agar (selects against gram-negatives)
Differential medium
Differential appearance:
Differential media allow the growth of more than one microorganism of interest but with
morphologically distinguishable colonies.
Note that almost any medium containing a specific substrate and well tailored indicator can be used
as a differential medium.
Examples of differential media include:
Mannitol Salts Agar (mannitol fermentation = yellow)
Blood Agar (various kinds of hemolysis)
MacConkey Agar (lactose fermentation = yellow)
Eosin-Methylene blue agar (various kinds of differentiation)
Enrichment culture
Technically enrichment culture differs from selective medium in not out and out suppressing the
growth of non-enriched microorganisms (e.g., no selective poisons are employed).
Enrichment culture is used to increase the relative concentration of certain microorganisms in the
culture prior to plating on solid selective medium.
Unlike selective media, enrichment culture is typically used as broth medium.
Pure culture technique
Pure culture technique is a method of culturing microorganisms in which all of the individuals in a
culture have descended from a single individual. This is done so as to: (i) inhibit evolutionary
change within cultures (ii) allow the characterization of types of microorganisms without the
confounding presence of other different types of microorganisms
Colony isolation:
The basis of pure culture technique is the isolation, in colonies, of individual cells, and their
descendants, from other colonies of individuals.
This is usually done by culturing methods employing petri dishes such as:
 streaking
 pouring
 spreading

The Growth Curve

This is a curve that describes the entire growth and growth pattern. It is the growth of
microorganism reproducing by binary fission, plotted as the logarithm of the number of viable
cells versus the incubation time.
The growth curve has four phases; the lag phase, the exponential phase, the stationary phase
and the death phase.

Growth is shown as L =log(numbers) where numbers is the number of colony forming units per
ml, versus T (time)

Phases
Bacterial growth curve/kinetic curve
The Lag phase
This is a phase in which there is no increase in the cell number of a microbial population freshly
introduced into a fresh culture medium. In this phase, there is no cell division and growth.
However, the cell is metabolically active synthesizing new components. Lag phase before cell
division begins may be necessary for these reasons:
 The cell may be old and depleted of ATP, essential growth factors and ribosome which
the cell synthesis at this phase or stage.
 The new medium may be different from the one the microorganism was growing
previously; therefore, the cells synthesize new enzymes to be used in the new medium.
 The cell is acclimatizing to a new environment
 The cells may be injured and require time to recover.

The period of the lag phase varies depending on the condition of the microorganisms and the
nature of the medium. This phase may be long, if
 The inoculum is from an old culture or one that has be refrigerated
  If the new medium is chemically different from the old one from which the organism
was taken. However, if a young actively growing culture is transferred from a fresh
medium of the same composition, the lag phase will be short or absent.

Exponential phase
This phase is also known as the log phase. The log phase is a period in which microorganisms
are actively growing and dividing at the maximal rate possible given their genetic potentials, the
nature of the medium and the condition under which they are growing. The rate of growth is
constant during this phase because the microorganisms are actively dividing and doubling in
number at regular interval. It is this period that the generation time of the organism is
determined.
The Stationary phase
This is a phase in which population growth ceases and the growth curve becomes horizontal. In
this phase, the total number of viable microorganism remains constant. This may result from a
balance between cell division and cell death or the population may simply cease to divide but
remain metabolically active.
Factors responsible for stationary phase when a required nutrient is exhausted include;
 Nutrient limitation: If an essential nutrient has been used up, e.g O 2 or carbon source
becomes unavailable to the microorganism, population growth will cease.
 Accumulation of toxic waste products: e. g Streptococci can produce too much lactic acid
and other organic acids from sugar fermentation that their medium becomes acidic and
growth is inhibited
 When a critical population level has been reached: This will prevent further dividing and
doubling of the cells/ when physical conditions do not permit a further increase in
population size.

Once the stationary growth phase is reached, there is no further net increase in bacterial cell
number.
Death phase
This is a phase in which the number of viable cells begin to decline. During this phase, the
number of living cells decreases because the rate of cell death exceeds the rate of new cell
formation. The depletion of essential nutrients and the accumulation of metabolic products such
as acids contribute to the death rate.
Tutorial Questions
(i) In what phase of the growth curve are cells dividing in a regular and orderly
process.
(ii) Why do cells enter the stationary phase

Measurement of microbial growth

Growth in microbiology refers to the magnitude of the total population i.e increase in the number
of cells as a consequence of cell growth and division. All growth studies of microorganisms are
population studies involving very large number of organisms. Measurement of microbial growth
helps to determine growth rate and generation time. Population growth is measured by following
changes in the number of cells or changes in some levels of cellular components. These could be
done by measuring the total cell number or by measuring the cell mass. Bacterial growth can be
determined quantitatively by numerous techniques based on the following types of measurement.
a. Cell count: It could be achieved directly by microscopy or by using electronic particles
counter or indirectly by colony count.
b. Cell mass: It can be done directly by weighing or by measurement of cell nitrogen or
indirectly by turbidity.
c. Cell activity: it can be done indirectly by relating the degree of biochemical activity to
the size of the population.

(A) Determination of cell number (cell count)

(i) Indirect method
 Plate count method: These methods involve plating serial dilutions of a suspension of
microorganisms unto a suitable solid growth medium after a period of incubation (in
which single cells multiply to form visible colonies), the number of colonies are counted
and enumerated. These methods are referred to as viable counting methods because they
count only those cells that are alive and able to reproduce. Two commonly used methods
are the spread plate and pour plate techniques
Limitation of this method
(a) Organisms could be affected by the brief heating (pour plate technique i.e assumption of
temperature i. e 45oC of the agar).
(b) No single medium is available which will allow the growth of all bacteria, thus only bacteria
that can grow in the medium used and under the condition of incubation provided will be
counted.
(c) Each viable organism capable of growing under the culture condition provided may not
necessarily give rise to one colony.

Advantage
(a) Very sensitive
(b)Allowance for positive identification of the organisms counted as the colony formed could
serve as inoculums for a pure culture and can be identified taxonomically.
(c) Several kinds of organisms can be counted in a mixture since different organisms produce
colony of different shape, size, texture and color.

 Membrane filter count

The set up contain a conical flask, a filter and a vacuum pump. After filtration the filter is
transfer into an already prepared medium. This is based on the use of molecular or membrane
filter of known uniform porosity and of predetermined size sufficiently small to trap
microorganisms after which the filter is placed in a special plate containing a pad saturated with
the appropriate medium. Special media and dyes can be used to make the detection of certain
types of organism easier. During incubation, the organism grows into colonies which appear on
the membrane surface.
(ii) Direct Method
(1). By direct microscopic count. The size of a microbial population may be measured by
counting the number of each individual cell with the microscope. A procedure known as direct
microscopic count. The small size of most microbes and their large population density allow the
use of special chambers such as petroff – Hausser or Heamocytometer chamber.
Limitation of the method
 Tedious and therefore impractical for large number of samples
  Not very sensitive because at least 10 6 bacteria cells per ml must be present before a
single cell would be seen in the microscope field.
 Living cells cannot be distinguish from dead cells
Advantage
 Morphology of the bacteria can be observed as they are counted.
 Very dense suspension can be counted if they are diluted appropriately
(iii). Electronic enumeration of cell numbers. The bacteria suspension is placed inside an
electronic particle counter and passed through a tiny orifice 10 - 30µm in diameter. This orifice
connects the two compartments of the counter to contain an electrically conductive solution.
Electrical signal generated as each bacterium passes through the orifice is counted.
Limitation
It does not determine viable cells, it only determines total cells.
(B) Determination of Cell Mass
The mass of the population rather than the number of cells present can be measured directly by
determining either the dry or the wet weight of sample taken from the population or by
centrifugation or by measuring the cell Nitrogen content.
(i) Determination of the dry or wet weight of cells
Advantage
a. Accurate and reliable way of measuring growth especially for research work
Limitation:
a. Viable cells cannot be determined
b. If is only useful when dealing with dense population.
(ii) Determination of Cell / Protoplasmic mass by centrifugation
This centrifuge is employed in the separation of two or more substances of different densities in
an aqueous medium by a force called centrifugal force. This principle is also applied to the
separation of microorganisms from its suspension medium. It therefore become suitable if no
component of the suspending medium can precipitate along with microbial cells.
(iii) Measurement of the total Nitrogen content
This can be determined micro kjeldahl method of which many procedures are available.
Limitations
a. Too laborious.
 b. It can be performed only on specimen free of all other sources of Nitrogen.
(iv) Estimation of Protein content in Microorganisms
This is based on the measurement of the metabolic activity of the organisms. The protein content
can be a measure for other substance. If the ratio of such substance to protein is known, for
example the protein content of most bacteria is about 50% of their dry weight depending on the
physiological condition of the organism. The Burette reaction and the Lowry et al method are
two photometric methods commonly used for this estimation.
(v) Turbidimeteric Method
The method is very simple in principle the more the number of cell in the suspension the greater
the turbidity or cloudiness. Evenly suspended microorganisms in liquid have the power to scatter
light passing through them. The amount of light absorbed and scattered is proportional to the
mass of cell in the light part. This ability of microorganisms to scatter light may be measured by:
a Simply comparing with standard turbidity tubes such as Brown’s opacity tubes.
b Special photoelectric devices which include Nephelometer which is used for direct
measurement of amount of light scattered. Colorimeter and Spectrophotometer could also
be used and they are used for measuring the amount of light lost from the beam by
scattering. In photoelectric colorimeter and spectrophotometer, light intensity is measured
by photoelectric response which can be made directly proportional to the quantity of light
falling on the photoelectric cell. The photo electric cell then transmits the light which is
recorded on the galvanometer.
Advantages of Turbidimetry method
a. It is the simplest of all.
b. A rapid method for following growth.
Limitations
a. Culture must be dense enough as total cell contribute to the turbidity.
b. It may not be possible to measure culture grown in deeply coloured media or cultures that
contain suspended material other than bacteria.
c. Dead and living cells contribute to the measurement.
d. The method is subject to errors if the cell grows in clumps.
 (C) Determination of Growth by Metabolic Activity
The principle is that the amount of reaction under specified conditions and during a fixed period
of time is directly proportional to the magnitude of the bacteria population e.g production of
organic acid from glucose fermentation, the measurement of acid or any other end product is a
very indirect approach to the measurement of growth. MPN (most probable number) is used for
estimating coliform bacteria in water, sewage, milk and milk product.
Factors Influencing the Growth of Microorganisms
 The rate of microbial growth and death are greatly influenced by environmental factors or
parameters.
 Some environmental conditions favor rapid microbial growth while others do not permit
bacterial reproduction.
 Understanding the influence of environmental factors on microorganisms help in the
control of microbial growth and the study of ecological distribution of microorganisms.
 These factors include; temperature, solute and water activity, pH, oxygen level, pressure,
radiation.

Temperature
Temperature is the most important factor affecting the growth and survival of microorganisms.
At either too cold or too hot temperature, microorganisms will not be able to grow or may even
die. For every microorganism, there is a minimum temperature below which growth is not
possible, an optimum temperature at which growth is most rapid and a maximum temperature
above which growth is not possible. These three temperatures are called the cardinal
temperature and are characteristics for any given microorganism. The temperature range at
which organisms grow best is called optimum temperature. On the basis of temperature
relationship, microorganisms are broadly divided into three groups:
• The psychrophiles : Phychrophiles grow optimally at 0-5°C,
• The mesophile: Mesophiles at temperatures between 20-45°C and
• The thermophiles: Thermophiles grow optimal at or above 55°C,
Oxygen requirement
The importance of oxygen to the growth of an organism correlate with its metabolism especially
with the processes it uses to conserve the energy supplied by its source. However,
microorganisms vary, remarkably in the amount of oxygen required for optimum growth Based
on the ability to grow in the presence or absence of oxygen. Microorganisms are classified as:
 Aerobes: are dependent on the presence of oxygen. They use and require oxygen as
electron acceptor. They have respiratory enzymes and lack the capacity for fermentation.
These are organisms that are able to grow in the presence of atmospheric oxygen. For
example, Pseudomonas and some Bacillus
 Anaerobes are sensitive to oxygen. Obligate or Strict Anaerobes grow in the absence of
atmospheric oxygen. They do not need oxygen in their nutrient. In fact, oxygen is a toxic
substance which either kills or inhibit their growth Obligate anaerobic procaryotes may
live by fermentation, anaerobic respiration. For example, Clostridium pasteurianum,
Bacteriodes
 Facultative anaerobes which can grow either in the presence or in the absence of
oxygen.
 Microaerophilic organisms need a lower concentration of oxygen in their
environment for growth. These organisms are damaged by normal atmospheric level
of oxygen (20%) and require O2 level below the range of 2 to 10% for growth e.g
Campylobacter

pH or Acidity

pH is a measure of the hydrogen ion activity of a solution and is defined as the negative
logarithm of the hydrogen concentration The pH scale extends from pH 0 to 14 and each pH
unit. It represents a tenfold change in in hydrogen ion concentration. The range of pH over which
an organism grows is defined by three cardinal points: the minimum pH, below which the
organism cannot grow, the maximum pH, above which the organism cannot grow and the
optimum pH at which the organism growth best. Based on pH growth range and pH growth
optimumwe have the following groups of microorganisms.

 Acidophiles: These organisms grow at pH range of 3. 0- 5. 0.

 Neutrophiles: These organisms grow best at neutral pH. Growth optimum betwwen 6.0 –
8.0
 Alkalophiles: Growth optimum between 8.0 – 11.5.
Moisture: Water is needed for the growth and metabolism like glycolysis and protein synthesis.
Various nutrients must be in a soluble form to facilitate diffusion into the cell. In the absence of
the water, some microorganisms will form spore to continue its survival.

Radiation:

Sunlight is the major source of radiation on the earth. It includes visible light, ultra violet
radiation, infrared ray and radio waves. Visible light is a most conspicuous and important aspect
of our environment. Most life is dependent on the ability of photosynthetic organisms to trap the
light energy of the sun. Most forms of electromagnetic radiation are very harmful to
microorganisms. This is particularly true of ionizing radiation, radiation of very short wavelength
and high energy, which can cause atom to lose electrons (ionize).

Tutorial Questions

(i) Enumerate the various methods of measurement of microbial growth

(ii) What are the advantages and disadvantages of the viable plate count method
(iii) What are cardinal temperatures
(iv) Describe the types of oxygen relationship seen in microorganisms.

Control of microorganisms: Sterilization and disinfection

Most microorganisms are beneficial to human beings; however, many have undesirable
consequences. They cause food spoilage and many diseases to plants, animal and human. In
microbiology, contaminants are undesirable or unwanted microbes present at a given place and
time. Therefore, it is imperative to control and inhibit the growth and activities of harmful or
undesirable microorganisms which are capable of causing diseases and contaminating water,
food and substances used.
Sterilization is the process that destroys or removes viable microorganisms including viruses
from an environment. Any material that has been subjected to the process is said to be sterile.
Sterility is the term used in relation, especially to microorganisms to describe the absence of all
life forms in an environment, surface of objects, or in an object which may be food, medical,
pharmaceutical products. Thus, an object is said to be sterile when it is free from all forms of
life. A sterile object is totally free of viable microorganisms, spores and other infectious agents.
 The method of sterilization used usually depends on the nature of the material to be sterilized.
The methods commonly used are classified as
 Physical control methods
 Chemical control methods.

STERIALIZATION

PHYSICAL CHEMICAL

HEAT FILTRATION RADIATION

DRY HEAT MOIST HEAT IONISATION NON-IONISATION

Physical methods of controlling microbial growth (Sterilization)

Heat and other physical agents are normally used to control microbial growth and sterilize
objects. The most frequently employed physical agents are heat, filtration and radiation
 heat,
 filtration and
 radiation.
(A) Heat Sterilization: Heat is the most commonly used physical sterilization method for
controlling and destroying microbial growth on materials which are resistant to heat
damage. Heat sterilization could be employed in different forms such as dry heat and moist
heat.
  Heat can kill microorganisms by denaturing the enzymes which prevent them from
multiplying.
 At temperature exceeding the maximal growth temperature, the death rate exceeds the
growth rate.
 Factors that determine the effectiveness of heat sterilization include the temperature and
duration of the heat treatment, and whether the heat is moist or dry
(i) Dry Heat Sterilization
This is a method of heat sterilization in which the objects or materials are sterilized in the
absence of water. Many objects are best sterilized in the absence of water by dry heat
sterilization. Dry heat is not as versatile or and widely used as moist heat but it has several
important sterilization applications. Dry heat kills the microorganisms by the oxidation of the
cell constituents and denaturation of proteins. It is extremely useful for materials that must
remain dry after sterilization. Examples are
 incineration (Red heat sterilization is done by using the Bunsen burner flame) for
example; inoculating loops used in the laboratory during the culturing of bacteria can be
sterilized in a small bench top incinerator.
 hot air Oven The oven at a temperature of 150 to 160oC for 2 to 3 hours can also be used
to sterilize glass wares such as pipettes, and test tubes in laboratories. This method of
sterilization has some definite advantages. It does not corrode glassware and metal
instruments as moist heat does and it can be used to sterilize powders, oils and other
similar items. However, it is very slow and less effective than moist heat. For example,
the spores of Clostridium botulinum, the organism that causes botulism are killed in 5
minutes at 121oC by moist heat but only after two hours at 160oC with dry heat.
 flaming after dipping into methylated spirit or ethanol. This is used in the sterilization
of such materials as scalpels, spatula, straight wires, loops as well as microscope slides
and other materials which are needed for immediate use but can withstand direct heating.
(ii) Moist Heat Sterilization

Moist heat readily destroys viruses, bacteria and fungi. Moist heat kills by degrading nucleic
acid and denaturing enzymes and other essential proteins. It also disrupts cell membranes.
Exposure of microorganisms to boiling water will destroy vegetative cells and eukaryotic spores.
However, this temperature will not kill bacterial endospores. In order to destroy bacterial
endospores moist heat sterilization above 100 oC using saturated steam under pressure is done.
Steam sterilization is carried out with an autoclave. Sterilization is done in the presence of steam
using various approaches. This method is used to sterilize liquids and materials which cannot
withstand dry heat and get easily charred. This method is effective as it sterilizes at low
temperature compare to dry heat, penetrates more quickly and does not dehydrate. It is mostly
used to sterilize gloves, masks, aprons, glass wares, instruments, media, water etc. The four ways
in which moist heat is employed to control microbes are:
 Steam under pressure- by autoclaving
 Non – pressurized steam – Tyndallization.
 Boiling water and
 Pasteurization

Sterilization with steam under pressure: This method is also known as autoclaving.
Sterilization of media, glass wares, instruments and water is done in an autoclave at 121 oC, 15psi
for 20min. The autoclave is an instrument which enables materials to be steamed at pressure
above the atmospheric pressure. To destroy bacteria endospore, moist heat sterilization must be
carried out at temperature above 100oC and thus requires the use of saturated steam under
pressure. This method is more effective than boiling as its uses pressure to raise the temperature
above the boiling. It even kills the most resistant spores of stearothermophilus .
Non pressurized steam: Selected substances that cannot withstand the high temperature of the
autoclave can be subjected to intermittent sterilization. This is useful for materials which get
damaged when subjected to temperature above 100oC. Intermittent sterilization also known as
Tyndallisation is a process of sterilizing by steam at 100oC for 30-60 minutes each of a three
successive days. In between steaming the liquid may be incubated at optimum temperature but is
usually kept at room temperature. The first exposure kills the heat sensitive vegetative cells but
not the spores. However, between the steam temperature (i.e. during incubation), the spores
germinate to produce vegetative forms which are then killed by subsequent steaming.

Boiling water: A simple boiling water bath or chamber can quickly decontaminate items in
clinic and home. Materials may be boiled in water bath at 100 oC for a specific period of (10-20
minutes). This is sufficient to kill vegetative microbial cells but not thermo-philes and
endospores. However, because a single processing at 100 oC will not kill all resistant cells, this
method can be relied on only for disinfection and not for sterilization

Pasteurization: The process whereby heat- labile materials such as milk, fruit juices, beer and
wines are subjected to moderate heat for a given period, thereby killing the potential agents of
infections and spoilage while retaining the liquid flavor, food value and prolong the storage life
is called Pasteurization. The process was first introduced by Luis Pasteur who used a
temperature of 50-60oC in killing yeast and bacteria capable of spoilage of wine. At a
temperature of 62oC for 30 minutes non -forming pathogenic bacteria are killed by pasteurization
however, thermoduric and spore- forming organisms are able to resist pasteurization effects.

(B) Filtration
Filtration is an effective method to remove microbes from air and liquid. In practice a fluid is
passed through a filter with openings large enough for the liquid to pass through but too small for
microorganisms to pass through. Filtration operates through exclusion rather than destruction of
microorganisms. That is, rather than directly destroying the contaminating microorganisms, the
filter simply removes them. Sterilization by filtration is used to prepare liquids that cannot
withstand heat including serum and other blood products, vaccines, drugs, vitamins, IV fluids
enzymes and media. Filtration is gradually replacing pasteurization in some cases, as it does not
cause any damage. Eg. Cold filtered beers. It is safe for the user and is employed for sensitive
liquids and gases as well. The filters are heat sterilized before use. The three types of filter
currently in use are.
(i) Depth filter
(ii) Membrane filter and
(iii) Nucleation track (Nucleopore filters)
(C) Radiation
Many forms of electromagnetic radiation are very harmful to microorganisms. Microwaves,
ultraviolet (UV) radiation, X-rays gamma rays (Y-rays) and electrons can effectively reduce
microbial growth if applied in the proper close. This is a low temperature method which can be
employed to sterilize materials or products to which heat sterilization could cause unacceptable
damage. Radiation denatures DNA. Sterilization by radiation can be by ionization and non-
ionization. However, because of the costs and hazards associated with radiation equipment, this
type of sterilization is limited to large industrial applications or specialized facilities. The use of
radiation sterilization practice has not been readily accepted in some countries because of fears
of possible radioactive contamination, alteration in nutritional value, production of toxic or
carcinogenic products, and perceived “off” tastes in irradiated food.
Ionization: Ionization radiation can include gamma rays and beta rays. They usually possess
high penetration power and are highly lethal to microorganisms (vegetables and spores). The
137
common method is to use gamma rays from a radioactive source such as Caesium and
60
Cobalt. Ionization radiation is useful in the sterilization of disposable goods such as plastic
petri-dishes and syringes as well as other medical instruments. However, ionizing radiation is not
always effective against viruses.
Non-Ionization: Ultraviolet (UV) ray do not penetrate surface and if they do only poorly. Hence
ultra violet radiation, although highly lethal to vegetative cells has little or no effect on spores. It
is present in natural daylight but can be obtained artificially from special mercury vapor lamps.
They are very useful in the sterilization of surfaces, air and inoculating chambers.
Chemical method of controlling microbial growth
Physical agents are generally used to sterilize objects. Chemicals on the other hand, are often
employed in disinfection and antisepsis. A large number of chemical compounds have the ability
to inhibit the growth and metabolism of microorganisms or to kill them. They are called
antimicrobial agents. These different chemicals are available for use as disinfectants and each
has its own characteristics and mode of action, advantages and disadvantages.
Chemicals are also employed to prevent microbial growth in foods and certain chemicals are
used to treat infectious disease. Chemicals bring about denaturation of proteins by hydrolysis,
oxidation or by attachment of atoms or reactive groups, heavy metals etc. They degrade the
cell wall, affect nucleic acid structure and hinder metabolism.
Antimicrobial chemicals occur in the liquid, gaseous and even solid states. They serve as
disinfectants, antiseptics, sterilants (chemicals that sterilize) degermers or preservatives
(chemicals that inhibits the deterioration of substances). In most cases, solid or gaseous
antimicrobials are dissolved in water, alcohols or a mixture of two to produce a liquid solution.
This section examines the term disinfection, the characteristics of an ideal disinfectant and
different chemical groups used as disinfectants.
Disinfection
 Disinfection is the killing, inhibition or removal of organisms that may be capable of
causing diseases.
 It is the process of destroying infectious agents.
 Disinfectants are antimicrobial agents, usually chemicals, that are applied to the surface
of non- living or inanimate objects to destroy microorganisms that are living on the on
the objects. Disinfection may not lead to the total removal of microorganisms because
viable spores and a few microorganisms may remain.
 Different disinfectants have different target ranges, not all disinfectants can kill all
microorganisms.
 Antimicrobial bleach (sodium hypochlorite) solution, for example, is a disinfectant used
to clean and disinfects food preparation areas.
Antimicrobial Agents
An antimicrobial agent is a natural or synthetic chemical that kills or inhibits the growth of
microorganisms. Agents that kill organisms are called -cidal agents, with a prefix indicating the
type of microorganisms killed. Thus, they are called bactericidal, fungicidal and viricidal
agents because they kill bacteria, fungi and viruses, respectively. Agents that do not kill but only
inhibit growth are called - static agents. These include bacteriostatic, fungistatic and viristatic
compounds.
Characteristics of an Ideal Antimicrobial Agent or Disinfectant
 It should have a broad spectrum of antimicrobial activity i.e. it must be effective against a
wide range of infectious agents such as gram positive and Gram negative bacteria, acid
fast bacteria, bacterial endospores, fungi and viruses.
 It must be active even at low concentration.
 It must be active in the presence of organic matter.
 Non-toxicity to human and other animals. It should be toxic to the infectious agent.
 It must be non-corroding and non-staining.
 It must be stable upon storage.
 Odorless or with pleasant smell.
 It must be soluble in water and lipids for proper penetration of microorganisms.
  It must be uniform in composition so that active ingredients are present in each
application.
 It must have a low surface tension so as to penetrate cracks in surfaces.
 It must be readily available.
 It must be relatively inexpensive.
Factors for the selection of a chemical agent
The major factors that need to be considered in the process of selecting the most appropriate
chemical agent for a specific practical application are:
 The nature of the material to be treated: e.g. a chemical agent used to disinfect
contaminated utensils might be quite unsatisfactory for application to the skin.
 Types of microorganisms: Chemical agents are not all equally effective against bacteria,
fungi, viruses, and other microorganisms. Spores are more resistant than vegetative cells.
Differences exist between Gram-positive and Gram-negative bacteria.
 Environmental condition: Such as temperature, pH, time, concentration, and presence of
extraneous organic materials, may all have a bearing on the rate and efficiency of
antimicrobial action.
Major groups of chemical antimicrobial agents: The commonly used chemical are;
 Phenol and phenolic compounds
 Alcohols
 Halogens
 Heavy metals and their compounds
 Dyes
 Detergents
 Quaternary ammonium compounds
 Aldehydes
 Gaseous agents