Conceptual of Microbiology

Taxonomy and Diversity

Classification
Impact of Microorganisms
The Scope of Microbiology
Varying opportunities
History
Microscope
• Microbiology is a fascinating field that deals with studying microorganisms, their
diversity, structure, functions, and interactions with other living organisms.
• Microbiology is the study of the biology of microscopic organisms - viruses,
bacteria, algae, fungi, slime molds, and protozoa.
• Microbiology is the study of microbes. Microbes, also called micro-organisms, are
a group of organisms that are too small to be seen with the naked eye.
• They are being of unicellular (single-celled), multicellular (consisting of complex
cells), or acellular (lacking cells)
• Microbes that cause disease are called pathogens.
• Human pathogens account for less than 1% of microbial species
• There are several microbes, including bacteria, archaea, protozoa, fungi, algae,
lichens, slime molds, viruses, and prions. Most of these organisms can survive
outside a host in the air or soil, except viruses, which can only survive briefly
outside their host cells.
• Taxonomy is the classification, nomenclature, and identification of microbes
(algae, protozoa, slime molds, fungi, bacteria, archaea, and viruses). The
naming of organisms by genus and species is governed by an international
code.
• Microbial diversity is defined as the variability among living organisms.
The main key to microbial diversity on Earth is due to evolution.
• The structural and functional diversity of any cell represents its evolutionary
event which occurred through the Darwinian Theory of natural selection.
• Natural selection and survival of the fittest theory are involved in the
microorganisms.
• This includes diversity within species, between species, and of ecosystems.
Micro-organisms may be classified into the following large biological
groups:
• Algae
• Protozoa
• Slime molds
• Fungi
• Bacteria
• Archaea
• Viruses.
Impact of Microorganisms:
Microbes are essential for life:
The majority of microorganisms help maintain the balance of life in our
environment.
• Marine and freshwater microorganisms form the basis of the food
chain in oceans, lakes, and rivers.
• Soil microbes help break down wastes and incorporate nitrogen gas
from the air into organic compounds, thereby recycling chemical
elements among soil, water, living organisms, and air.
Certain microbes play important roles in photosynthesis, a food and
oxygen-generating process critical to life on Earth.
Humans and many other animals depend on the microbes in their
intestines for digestion and the synthesis of some vitamins that their
bodies require, including some B vitamins for metabolism and vitamin
K for blood clotting.
Microorganisms also have many commercial applications.
• They are used in the synthesis of such chemical products as vitamins,
organic acids, enzymes, alcohols, and many drugs. For example,
microbes are used to produce acetone and butanol, and the vitamins
B2 (riboflavin) and B12 (cobalamin) are made biochemically.
• The process by which microbes produce acetone and butanol was
discovered in 1914 by Chaim Weizmann, a Russian-born chemist
working in England.
• The food industry also uses microbes in producing, for example,
vinegar, sauerkraut, pickles, soy sauce, cheese, yogurt, bread, and
alcoholic beverages.
• Only a minority of microorganisms are pathogenic
(disease-producing), practical knowledge of microbes is necessary for
medicine and the related health sciences.
• Various medical conditions with drugs produced by microbes.
• we dust our plants with insecticides of microbial origin.
• we use microorganisms as tiny factories to churn out various industrial
chemicals and plastics.
• We wash our clothes with detergents containing microbe-produced
enzymes.
The Scope of Microbiology:
• To study the natural history of microbes, it also deals with every
aspect of microbe-human and microbe-environmental interactions.
• These interactions include genetics, metabolism, infection, disease,
drug therapy, immunology, genetic engineering, industry, agriculture,
and ecology.
Microbiology is both a basic and applied science.
Specific types of organisms:
• Virologists (those that study viruses);
• Bacteriologists (those that study bacteria);
• Mycologists (those who study fungi);
• Protozoologists (those who study protozoa);
• Phycologists or Algologists (those that study algae).
Activities or characteristics of these microorganisms:
• Microbial cytology (those that study microbial cell components);
• Microbial morphology (those that study the structure of microbes);
• Microbial physiology (those that study the life processes of microbes);
• Microbial ecology (those that study the activities of microbes in their
habitat/niche);
• Microbial genetics and molecular biology (those that study microbes at the
molecular level);
• Microbial taxonomy (those that are concerned with the identification,
classification, and naming of microbes).
Applied focus:
• Applied microbiology is the application of microbiological principles and
techniques in the production of economically useful products/services and for
solving societal problems. It is the exploitation of microorganisms for the
production of a specific product including medicines, food, fuel, and beverages.
Different areas of applied microbiology:
• Industrial microbiology (the area that exploits microbes for use in industrial
processes such as in waste water treatment, bioremediation and fermentation
processes);
• Medical microbiology (the area that studies pathogenic microbes and the role of
microbes in human diseases);
• Pharmaceutical microbiology (the area which studies microbes that are related to
the production of antibiotics, enzymes, vitamins, vaccines, and other
pharmaceutical products).
• Agricultural microbiology (the area which studies agriculturally
relevant microorganisms; and it encompass soil microbiology and
plant microbiology/pathology);
• Food and Dairy microbiology (the area which study microorganisms
that cause food spoilage and food borne diseases; and it encompass the
application of microorganisms to produce foods);
• Environmental microbiology (the area which study the function and
diversity of microbes in their natural environments). Other scope of
microbiology such as aero-microbiology and geo-microbiology also
exist.
Varying opportunities:
• Biomedical sciences including medicine where the help and assistance of
microbiologists are required in the development of methods for the prevention,
detection, diagnosis and treatment of infectious diseases which are caused by
pathogenic microorganisms.
• Research and developments in the areas of biotechnological, pharmaceutical,
biochemical, medical, and chemical industries.
• Important roles in the public health sector, university education and their research
centers, the government, and the food and beverage industries – where the
principles of microbiology are applied to solve several problems of man, animals,
and the environment.
• Human health and welfare, especially through the production of medicines and
vaccines used for the treatment and prevention of infectious diseases in the human
population.
Microbiology has numerous practical uses in industry and medicine. Some
prominent areas that are heavily based on applications in microbiology are as
follows:
• Immunology studies the system of body defenses that protects against infection. It
includes serology, a discipline that looks for the products of immune reactions in
the blood and tissues and aids in diagnosis of infectious diseases by that means,
and allergy, the study of hypersensitive responses to ordinary, harmless materials.
• Public health microbiology and epidemiology aim to monitor and control the
spread of diseases in communities. The principal U.S. and global institutions
involved in this concern are the United States Public Health Service (USPHS)
with its main agency, the Centers for Disease Control and Prevention (CDC)
located in Atlanta, Georgia, and the World Health Organization (WHO), the
medical limb of the United Nations.
• The CDC collects information on diseases from around the United States and
publishes it in a weekly newsletter called the Morbidity and Mortality Weekly
Report.
• Food microbiology, dairy microbiology, and aquatic microbiology examine the
ecological and practical roles of microbes in food and water.
• Agricultural microbiology is concerned with the relationships between microbes
and crops, with an emphasis on improving yields and combating plant diseases.
• Biotechnology includes any process in which humans use the metabolism of living
things to arrive at a desired product, ranging from bread making to gene therapy.
• Industrial microbiology is concerned with the uses of microbes to produce or
harvest large quantities of substances such as beer, vitamins, amino acids, drugs,
and enzymes.
• Genetic engineering and recombinant DNA technology involve techniques that
deliberately alter the genetic makeup of organisms to mass-produce human
hormones and other drugs, create totally novel substances, and develop organisms
with unique methods of synthesis and adaptation. This is the most powerful and
rapidly growing area in modern microbiology.
Brief History of Microbiology:
• The study of microbiology cum microbes began and gained prominence with the
discovery of the microscope, a metallic piece of instrument that is used to see
microorganisms (invisible forms of life).
• The first person to postulate the existence of microorganisms was Aristotle in 4 B.
C. He suggested that living organisms are made up of cells.
• It was only until the 13th century that people realized that ground pieces of glass
provided a greater magnifying power. They were able to see tiny objects that they
could otherwise not see through their naked eyes.
• Roger Bacon postulated that invisible living creatures caused diseases.
• In 1530, Fracastoro of Verona coined the term syphilis to describe an outbreak that
ravaged Europe in the 1400’s when the returning French soldiers spread the
disease. He called the disease agent “seminaria morbi” (living germs) that spread
‘contagium vivum’ (via contact with an individual with the germ).
• In 1658, Athanasius Kircher defined the invisible organisms found in decaying
bodies, meat, milk, and secretions as worms.
• Even before microorganisms were seen, some investigators suspected their
existence and responsibility for disease. Among others, the Roman philosopher
Lucretius (about 98–55 B.C.) and the physician Girolamo Fracastoro (1478–1553)
suggested that disease was caused by invisible living creatures.
• The earliest microscopic observations appear to have been made between 1625
and 1630 on bees and weevils by the Italian Francesco Stelluti, using a microscope
probably supplied by Galileo.
• Robert Hooke (1635-1703) was the first to use the microscope to view the unseen
forms of life (particularly the fruiting bodies of molds), making him the first to
describe microorganisms.
• Hooke as shall be seen later in this section reported seeing plant and fungal
structures under his crude compound microscope whose lenses were unable to
view bacteria. His observations were drawn and recorded in his famous book titled
Micrographia. In 1665, the first drawing of a microorganism was published in
Robert Hooke’s Micrographia.
• Antonie van Leeuwenhoek (1632-1723), who is widely regarded as the father of
the field of microbiology, was the first microbiologist to see and describe bacteria.
i.e. To experiment on microscopic organisms (for example, bacteria).
• The first person to publish extensive, accurate observations of microorganisms
was the amateur microscopist Antony van Leeuwenhoek (1632–1723) of Delft,
The Netherlands. Leeuwenhoek earned his living as a draper and haberdasher (a
dealer in men’s clothing and accessories) but spent much of his spare time
constructing simple microscopes composed of double convex glass lenses held
between two silver plates.
• The microscopes of Leeuwenhoek could magnify objects about 200-300 times.
With his microscopes, Leeuwenhoek observed a variety of things like rainwater,
pond water, and scrapings from his teeth. He saw minute moving objects and
called them as “Little animalcules”, which we now know as protozoa, yeasts, and
bacteria. He made accurate sketches and communicated his findings to the “Royal
Society of London”. Thus, Leeuwenhoek was the first person to discover the
microscope and the presence of bacteria and spirochetes in the mouth.
EDWARD JENNER (1749-1823):
• Jenner was an English country physician, who discovered a safe and
efficient vaccination against smallpox. Which ultimately led to the
eradication of smallpox (Variola). Jenner observed that dairy workers,
exposed to occupational cowpox infection were immune to smallpox.
He proved experimentally that resistance to smallpox can be induced
by injecting cowpox material (Vaccinia) from disease pustules into
man (in 1796). Pasteur gave the general term “Vaccine” (Vacca = cow)
in honor of Jenner’s cowpox vaccine, to various materials used to
induce active immunity. Jenner published his findings in 1798 in a
pamphlet “An inquiry into the cause and effect of a variole vaccine”.
• The active ingredient in all vaccines is an antigen, the name for any
substance that causes the immune system to begin producing
antibodies. In a vaccine, the antigen could be either. Weakened or
killed bacteria or viruses.
LOUIS PASTEUR (1822-1895)
He was a Professor of Chemistry at the University of Lille, France. He is considered
as “Father of Microbiology”, as his contribution led to the development of
Microbiology as a separate scientific discipline. He proved the theory of
“Biogenesis” and disproved the “Theory of spontaneous generation”(Abiogenesis),
experimentally by using swan-necked flasks.
He worked on souring of wine and beer and found that this alcohol spoilage is due
to the growth of undesirable organisms, while the desirable microorganisms produce
alcohol by a chemical process called “Fermentation”. He showed that wine did not
spoil, if it is heated to 50-60°C for a few minutes. This method is called
“Pasteurization”, now widely used in dairy units, to kill pathogenic microorganisms
in milk.
He is a founder of “Germ theory of disease” as he visualized that diseases are
caused by microorganisms. In course of his research, he discovered the importance
of sterilization and discovered steam sterilizer, autoclave and hot air oven.
• He also established the importance of cotton wool plugs for protection of culture
media from aerial contamination.
• He differentiated between aerobic and anaerobic bacteria and coined the term
“anaerobic” to refer to the organisms that do not require oxygen for growth.
• He worked on “Pebrine”, a silk-worm disease caused by a protozoan and showed
that infection can be controlled by choosing worms free from the parasite for
breeding.
• He developed the process of “attenuation” during his work on “chicken cholera”
in fowls. He found that cultures which had been stored in the laboratory for
sometime would not kill the animals as fresh cultures did. This attenuation is now
used in protective vaccination against diseases.
• Pasteur showed that the anthrax disease in cattle and sheep is caused
by a bacterium. He cultivated anthrax organisms in sterile yeast water,
and showed that these cultures can produce disease when inoculated in
to healthy animals. He developed a live attenuated anthrax vaccine, by
incubation at 40-42°C, which proved to be useful in protecting animals
against anthrax.
• Pasteur developed a vaccine against rabies (Hydrophobia), which
made a greatest impact in medicine. He obtained the causative agent of
rabies by serial intracerebral passage in rabbits and the vaccine was
prepared by drying pieces of spinal chord.
• In 1888, Pasteur institute was established for mass antirabic treatment.
Pasteur gave the general term “Vaccine” (Vacca=cow) in honour of
Jenner’s cow pox vaccine, to various materials used to induce active
immunity.
• ROBERT KOCH (1843-1912):
• He was a German country Doctor who later became the Professor of Hygiene and
Director of the Institute of Infective Diseases in Berlin. He perfected many
bacteriological techniques and is known as the “Father of Practical Bacteriology”.
He discovered rod-shaped organisms in the blood of animals, that died of anthrax.
He experimentally obtained the anthrax organisms in pure culture on a depression
slide by inoculation of infected blood.
• He observed the multiplication of bacteria and spore formation. He injected these
spores into mice and reproduced the disease. He found that in certain conditions,
the anthrax bacillus forms spores, that can survive on earth for years. He passed
anthrax bacilli, from the blood of an infected animal, from one mouse to another
through twenty generations, and found that they bred true. He worked out its life
history.
• He introduced staining techniques. He prepared dried bacterial films (Smears) on
glass slides and stained them with aniline dyes to produce a better contrast under a
microscope. He discovered tubercle bacillus (Mycobacterium tuberculosis),
known as Koch’s bacillus. He injected tubercle bacilli into laboratory animals and
reproduced the disease, satisfying all of Koch’s postulates.
• He discovered Vibrio cholerae, the causative agent of cholera disease. He
developed pure culture techniques by introducing solid media. The use of
agar-agar obtained from dried sea weeds (Gelidium Sp.) in the preparation of solid
bacteriological media was first suggested by Frau Hesse, the wife of Koch’
student. This agar-agar is totally inert with no nutritive value, solidifies at 45°C
and melts at 90°C, and was found to be most suitable solidifying agent in the
preparation of culture media. Koch isolated bacteria in pure cultures on these solid
media. It revolutionized bacteriology.
• He discovered “Old Tuberculin”. Koch noted that when tubercle bacilli or its
protein extract was injected into a Guinea-pig already infected with the bacillus,
an exaggerated reaction took place and the reaction remain localized. This is
popularly called “Koch Phenomenon” and it is a demonstration of cell mediated
immunity. The tuberculin test is based on Koch’s phenomenon. He erroneously
thought that protein extracted from tubercle bacilli, called “Old tuberculin”, could
be used in the treatment of tuberculosis. Koch did a series of experiments to fulfill
the criteria laid by his teacher Henle to establish the causative role between a
particular microorganism and a particular disease.
 They are popularly known as Koch’s postulates (Henle-Koch’s Postulates).
They are :
1. A specific organism should be found constantly in association with the disease.
2. The organism should be isolated and grown in a pure culture in the laboratory.
3. The pure culture when inoculated into a healthy susceptible animal should
produce symptoms/ lesions of the same disease.
4. From the inoculated animal, the microorganism should be isolated in pure
culture.
5. An additional criterion introduced is that specific antibodies to the causative
organism should be demonstrable in the patient’s serum.
IWANOWSKY (1892)
• Dmitri Iwanowsky, a Russian botanist, occupies a pivotal position in
the history of virology. In 1866, Adeolf E. Meyer, a Dutch agricultural
chemist described a disease of tobacco called “Mosaic” and showed
that the disease could be transmitted to healthy plants through the sap
of the diseased plant. Iwanowsky (1892) demonstrated that this disease
was caused by an agent which could pass through the filter, which
withholds bacteria.
• He obtained the sap from infected leaves and passed it through a
bacterial filter, called chamberland candle filter, which retained all
bacteria and the filtered sap still retained infectivity when applied to
healthy leaves. Beijerinck (1898), a Dutch Microbiologist, showed that
this infectious agent could diffuse through an agar gel and that it was a
non-corpuscular “Contagion vivum fluidum” which he called “Virus”.
ALEXANDER FLEMMING (1881-1955):
• He was an English scientist worked at St. Mary’s hospital in London. Flemming was associated with
two major discoveries-lysozyme and penicillin. In 1922, he discovered lysozyme by demonstrating
that the nasal secretion has the power of dissolving or lysing certain kinds of bacteria. Subsequently,
he showed that lysozyme was present in many tissues of the body.
• In 1929, Flemming made an accidental discovery that the fungus Penicillium notatum produces an
antibacterial substance which he called penicillin. Flemming was culturing Staphylococci in
petridishes and some of his cultures were contaminated with a mold, subsquently indentified as
Penicillium notatum. Around the mold colony, there were clear zones, where Staphylococci
disappeared. Flemming attributed this to the production of an antibacterial susbstance by the mold.
• Flemming cultured the fungus Penicilium notatum in broth cultures, filtered the fungal mat and
obtained the penicillin in soluble form in the culture filtrate. In 1940, Howard Florey and Ernst Chain
demonstrated its antibacterial action in vivo. Working in U.S.A., they were able to produce large
quantities of penicillin in pure form. In 1945, Flemming, Florey and Chain shared the nobel prize in
physiology and medicine for the discovery of penicillin.
SELMAN WAKSMAN :
• Waksman's team discovered several antibiotics, including actinomycin, clavacin,
streptothricin, streptomycin, grisein, neomycin, fradicin, candicidin, candidin, and
others. Two of these, streptomycin and neomycin, have found extensive
application in the treatment of numerous infectious diseases. Streptomycin was the
first antibiotic that could be used to cure the disease tuberculosis. Waksman coined
the term antibiotics.
• He was the father of Byron Waksman, involved in Multiple sclerosis research.
Other little known contributions of Selman Waksman include anti-fouling paints
for the Navy, the use of enzymes in detergents, and the use of Concord grape root
stock to protect the French Vineyards from fungal infection.
Microscope
Definition:
• It is an instrument that deals with organisms too small to be seen
distinctly by the naked eye.
Characteristics:
• Resolution
• Contrast
• Magnification
• The human eye can resolve objects of the order of 0.1 mm,
• the light microscope can resolve objects on the order of 0.2 µm (200
nm) with a magnification of 1000.
• The transmission electron microscope, can resolve objects on the order
of 0.1nm (100 A ˚ units).
History of Microscope:
• 1000AD – The first vision aid was invented (inventor unknown) called a
reading stone. It was a glass sphere that magnified when laid on top of reading
materials.
• 1284 - Italian, Salvino D'Armate is credited with inventing the first wearable
eye glasses.
• 1590 – Two Dutch eye glass makers, Zaccharias Janssen and son Hans Janssen
experimented with multiple lenses placed in a tube. The Janssens observed that
viewed objects in front of the tube appeared greatly enlarged, creating both the
forerunner of the compound microscope and the telescope.
• 1665 – English physicist, Robert Hooke looked at a sliver of cork through a
microscope lens and noticed some "pores" or "cells" in it.
• 1674 – Anton van Leeuwenhoek built a simple microscope with only one lens
to examine blood, yeast, insects and many other tiny objects. Leeuwenhoek was
the first person to describe bacteria.
 • 18th century – Technical innovations improved microscopes, leading to
microscopy becoming popular among scientists. Lenses combining two types
of glass reduced the "chromatic effect" the disturbing halos resulting from
differences in refraction of light.
• 1830 – Joseph Jackson Lister: reduces spherical aberration or the "chromatic
effect" by showing that several weak lenses used together at certain distances
gave good magnification without blurring the image. This was the prototype for
the compound microscope.
• 1872 – Ernst Abbe wrote a mathematical formula called the "Abbe Sine
Condition". His formula provided calculations that allowed for the maximum
resolution in microscopes possible.
• 1903 – Richard Zsigmondy developed the ultra microscope that could study
objects below the wavelength of light. He won the Nobel Prize in Chemistry in
1925.
• 1932 – Frits Zernike invented the phase-contrast microscope that allowed for the
study of colorless and transparent biological materials for which he won the Nobel
Prize in Physics in 1953.
• 1931 – Ernst Ruska co-invented the electron microscope for which he won the
Nobel Prize in Physics in 1986. An electron microscope depends on electrons rather
than light to view an object, electrons are speeded up in a vacuum until their
wavelength is extremely short, only one hundred thousandth that of white light.
Electron microscopes make it possible to view objects as small as the diameter of an
atom.
• 1981 – Gerd Binnig and Heinrich Rohrer invented the scanning tunneling
microscope that gives three-dimensional images of objects down to the atomic level.
Binnig and Rohrer won the Nobel Prize in Physics in 1986.
Introduction to Light Microscope:
• As early as the 4th century AD, people had discovered the basic concept of an
optical lens. By the 13th century, they were already using glass lenses to improve
their eyesight and to magnify objects such as plants and insects to understand them
better.
• Light microscopy is a core technique in many areas of science and technology,
including life sciences, biology, materials sciences, nanotechnology, industrial
inspection, forensics and many more.
• Light microscopy is used to make small structures and samples visible by
providing a magnified image of how they interact with visible light, e.g., their
absorption, reflection and scattering.
• This is useful to understand what the sample looks like and what it is made of, but
it also allows us to see processes of the microscopic world, such as how
substances diffuse across a cell membrane.
Mechanism behind light Microscope:
• When light energy passes from one medium to another (i.e. AIR and GLASS)
the light rays are bent at the point of interface. This process is called refraction.
• The measure of how greatly a substance slows the velocity of light is called the
refractive index
• The direction and magnitude of bending are determined by the refractive
indexes of the two media forming the interface.
• As parallel light rays encounter a convex lens, they are slowed and bent toward
the normal path.
• The point at which these rays converge is called the focal point
• The distance between the center of the lens and the focal point is called the
focal length.
• The strength of a lens is directly related to its focal length. A lens with a short
focal length has a greater capacity for magnification than a lens with a longer
focal length.
PARTS:
• A microscope comprises two subsystems: an illumination system to illuminate the sample
and an imaging system that produces a magnified image of the light that has interacted
with the sample, which can then be viewed by eye or using a camera system.
• Early microscopes used an illumination system comprising sunlight that was collected and
reflected onto the sample by a mirror. Today, most microscopes use artificial light sources
such as light bulbs, light-emitting diodes (LEDs) or lasers to make more reliable and
controllable illumination systems, which can be tailored to a given application.
• In these systems, light from the source is typically collected using a condenser lens and
then shaped and optically filtered before being focused onto the sample. Shaping the light
is essential to achieve high resolution and contrast, and often includes controlling the
sample area that is illuminated and the angles at which light impinges on it.
• Optical filtering of the illumination light, using optical filters that modify its spectrum and
polarization, can be used to highlight certain features of a sample, to improve the
visibility of weak signatures or to observe a sample’s fluorescence.
• The imaging system collects illuminating light that has interacted with the sample
and produces a magnified image that can be viewed. This is achieved using two
main groups of optical elements:
• First, an objective lens that collects as much light from the sample as possible
• Second, an eyepiece lens which relays the collected light to the observer’s eye or a
camera system.
• The imaging system may also include elements such as apertures and filters that
select certain portions of light from the sample, for example to see only light that
has been scattered off the sample, or only light of a certain color or wavelength.
• As in the case of the illumination system, this type of filtering can be extremely
useful to single out certain features of interest that would remain hidden when
imaging all the light from the sample.
Simple Microscope:
• A single lens can be used as a magnifying glass which increases the
apparent size of an object when it is held close to the lens. Looking
through the magnifying glass at the object, we see a magnified and
virtual image of the object.
• This effect is used in simple microscopes, which consist of a single
lens that images a sample held clamped into a frame and illuminated
from below. This type of microscope can achieve a magnification of
typically 2-6 x, which is sufficient to study relatively large samples.
• However, achieving higher magnification and better image quality
requires the use of more optical elements, which led to the
development of the compound microscope.
Compound Microscope:
• A compound microscope is often referred to as a biological microscope.
The Parts & Function of a Compound Microscope:
• A compound microscope is a high-power (high magnification) microscope
that uses a compound lens system. A compound microscope has multiple
lenses: the objective lens (typically 4x, 10x, 40x, or 100x) is compounded
(multiplied)
• by the eyepiece lens (typically 10x) to obtain a high magnification of 40x,
100x, 400x, and 1000x.
• Higher magnification is achieved by using two lenses rather than just a
single magnifying lens.
• While the eyepieces and the objective lenses create high magnification, a
condenser beneath the stage focuses the light directly into the sample.
Bright-field Microsccopy:
• Bright field Microscopy is the simplest of all the optical microscopy illumination
techniques.
• Sample illumination is transmitted (i.e., illuminated from below and observed
from above) white light, and contrast in the sample is caused by absorbance of
some of the transmitted light in dense areas of the sample.
• The typical appearance of a bright field microscopy image is a dark sample on a
bright background, hence the name.
Components are necessary for Brightfield Microscopy:
• Light path
• Transillumination light source
• Condenser lens
• Objective lens
• An eyepiece, or ocular lens
• Focusing knobs to adjust the focus of the image
Working Performance:
• Bright field microscopy typically has low contrast with most biological samples as
few absorb light to a great extent.
• Staining is often required to increase contrast, which prevents use on live cells in
many situations.
• Bright field illumination is useful for samples with an intrinsic color, such as
chloroplasts in plant cells.
• Light is first emitted by the light source and is directed by the condenser lens onto
the specimen.
• The light from the specimen then passes through the objective lens. This lens is
often selected from among three or four and is the main determinant for the level
of magnification. It bends the light rays.
• The light rays then travel to the oracular lens or “eyepiece". This is often a 10X
magnification lens, meaning it magnifies the magnified image an additional ten
times. The image is then projected into the eye.
Advantages:
• The name "brightfield" is derived from the specimen being dark and contrasted by
the surrounding bright viewing field. Simple light microscopes are sometimes
referred to as bright field microscopes.
• Brightfield microscopy is very simple to use with fewer adjustments needed to be
made to view specimens.
• Some specimens can be viewed without staining and the optics used in the
brightfield technique do not alter the specimen.
• It is adaptable with new technology and optional pieces of equipment can be
implemented with brightfield illumination to give versatility in the tasks it can
perform.
Disadvantages:
• By using an aperture diaphragm for contrast, past a certain point, greater contrast
adds distortion. However, employing an iris diaphragm will help compensate for
this problem
• Bacteria have an optimum viewing magnification of 1000x.
• Brightfield microscopy has very low contrast and most cells have to be stained to
be seen; staining may introduce extraneous details into the specimen that should
not be present.
• Also, the user will need to be knowledgeable in proper staining techniques.
• Lastly, this method requires a strong light source for high magnification
applications and intense lighting can produce heat that will damage specimens or
kill living microorganisms.
Darkfield Microscope:
• This is similar to the ordinary 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 darkfield than with
brightfield microscopy.
• Dark-field microscopy is a technique that can be used for the observation of
living, unstained cells and microorganisms. In this microscopy, the specimen is
brightly illuminated while the background is dark.
Advantages of Dark-Field Microscopy
• Resolution by dark-field microscopy is somewhat better than bright-field
microscopy.
• It improves image contrast without the use of stain and thus does not kill cells.
• Direct detection of non-culturable bacteria present in patient samples.
• No sample preparation is required.
• It requires no special setup, even a light microscope can be converted to a dark
field.
Limitations of Dark-Field Microscopy
• Necessity to examine wet, moist specimens containing living organisms very
quickly, because visualization of the moving bacteria is essential to detection.
• The sample must be very strongly illuminated, which can cause damage to the
sample.
• Besides the sample, dust particles also scatter the light and appear bright.
• Sample material needs to be spread thinly, dense preparations can grossly affect
the contrast and accuracy of the dark field’s image.
Phase contrast microscopy
• Phase contrast microscopy is an optical microscopy technique that converts
phase shifts in the light passing through a transparent specimen to brightness
changes in the image.
• Phase shifts themselves are invisible but become visible when shown as
brightness variations.
• When light waves travel through a medium other than a vacuum, interaction
with the medium causes the wave amplitude and phase to change in a manner
dependent on the properties of the medium.
• Changes in amplitude (brightness) arise from the scattering and absorption of
light, which is often wavelength-dependent and may give rise to colors.
• Photographic equipment and the human eye are only sensitive to amplitude
variations.
• Without special arrangements, phase changes are therefore invisible. Yet, often
these changes in phase carry important information.
Working Principle:
• The basic principle to make phase changes visible in phase contrast microscopy is
to separate the illuminating background light from the specimen’s scattered light,
which makes up the foreground details, and to manipulate these differently.
• The ring-shaped illuminating light (green) that passes the condenser annulus is
focused on the specimen by the condenser. Some of the illuminating light is
scattered by the specimen (yellow). The remaining light is unaffected by the
specimen and form the background light (red). When observing unstained
biological specimen, the scattered light is weak and typically phase shifted by -90°
relative to the background light. This leads to that the foreground (blue vector) and
the background (red vector) nearly have the same intensity, resulting in a low
image contrast.
In a phase contrast microscope, the image contrast is improved in two steps.
• The background light is phase-shifted -90° by passing it through a phase shift ring.
This eliminates the phase difference between the background and the scattered
light, leading to an increased intensity difference between foreground and
background.
• To further increase contrast, the background is dimmed by a gray filter ring. Some
of the scattered light will be phase-shifted and dimmed by the rings. However, the
background light is affected to a much greater extent, which creates the phase
contrast effect
• The above describes negative phase contrast. In its positive form, the
background light is instead phase-shifted by +90°. The background light will
thus be 180° out of phase relative to the scattered light. This results in that the
scattered light will be subtracted from the background light to form an image
where the foreground is darker than the background.
Application:
• The standard light microscope has some limitations of low contrast that does not
give out a perfect image of the biological samples. To eradicate this limitation,
phase-contrast microscopy is used. Here, the biological sample gets illuminated
with good contrast and light.
• The samples used here, tend to execute the diffraction process which gives out the
changes in the various refractive indexes which reveal many structures of the
biological samples. However, in some cases, the refractive index differences are so
slim that the structures cannot be revealed. But with a perfect inverted
phase-contrast microscope, the images can be formed in the microscope’s
objective area.
Fluorescence Microscope:
• A fluorescence microscope is similar to a regular light microscope, but it has
several extra qualities that enhance its usefulness. The typical microscope
magnifies a sample using visible light (400-700 nanometers).
• On the other hand, Fluorescence microscopes use high-intensity light to
stimulate fluorescent organisms in a sample.
• This fluorescent species emits a longer wavelength, lower-energy light that
magnifies the image.
• Therefore, a fluorescence microscope is an optical microscope that studies
the properties of organic or inorganic substances by using fluorescence and
phosphorescence instead of or in addition to reflection and absorption.
• Fluorescence was first discovered in 1845 by Fredrick W. Herschel.
• However, the first working fluorescent microscope was developed by Oskar
Heimstaedt in 1911.
Parts of fluorescence microscope:
• Fluorescent dyes (Fluorophore): A fluorophore is a fluorescent
chemical compound that can re-emit light upon light excitation.
Fluorophores typically contain several combined aromatic groups, or
plane or cyclic molecules with several π bonds. Many fluorescent
stains have been designed for a range of biological molecules. Some
are fluorescent tiny compounds that bind a biological molecule.
Nucleic acid stains like DAPI and Hoechst, and phalloidin are
examples.
• A light source: Four main light sources are used, including xenon arc
lamps or mercury-vapor lamps with an excitation filter, lasers, and
high-power LEDs. Complex fluorescence microscopy techniques
require lasers, while wide-field epifluorescence microscopes use
xenon lamps, mercury lamps, and LEDs with a dichroic excitation
filter.
• The excitation filter: Typically, the exciter is a bandpass filter that transmits
only the wavelengths absorbed by the fluorophore, reducing the excitation
of other fluorescence sources and blocking excitation light in the
fluorescence emission band.
• The dichroic mirror: A dichroic filter or thin-film filter is a very accurate
color filter used to selectively pass light of a small range of colors while
reflecting other colors.
• The emission filter: The emitter is typically a bandpass filter that passes
only the wavelengths emitted by the fluorophore and blocks all undesired
light outside this band – especially the excitation light.
• By blocking unwanted excitation energy (including UV and IR) or sample
and system autofluorescence, optical filters ensure the darkest background.
Working Principle of Fluorescence Microscope:
• Most cellular components are colorless and cannot be clearly distinguished under
a microscope.
• The basic premise of fluorescence microscopy is to stain the components with
fluorescent dyes, also known as fluorophores or fluorochromes, which are
molecules that absorb excitation light at a given wavelength (generally UV), and
after a short delay emit light at a longer wavelength.
• To observe the sample through a fluorescence microscope, it should first be
labeled with fluorescent dyes/substances known as fluorophores. Higher energy
shorter wavelength lights (UV rays or blue light) generated from xenon arc lamp
or mercury vapor arc lamp passes through the excitation filter.
• The excitation filter allows only the short wavelength of light to pass through and
removes all other non-specific wavelengths of light. The filtered light is reflected
by the dichroic filter and falls on the fluorophore-labeled sample.
• The fluorochrome absorbs shorter wavelength rays and emits rays of longer
wavelength (lower energy) that pass through the emission filter. The emission
filter blocks (suppresses) any residual excitation light and passes the desired
longer emission wavelengths to the detector.
• Thus the microscope forms glowing images of the fluorochrome-labeled
microorganisms against a dark background. The background is dark to the
observer, as there is no visible light and only the labeled specimen (cells,
microorganisms, etc.) appear bright (fluoresce).
Types of Fluorescence Microscopes:
Fluorescence microscopy is one of the most used imaging modalities in molecular biology and living
specimens. To increase image contrast and spatial resolution, different types of fluorescence
microscopy have been developed. However, there are 4 main types of fluorescence microscopy:
a) Epifluorescence microscopes: It is the most common type of fluorescence microscope. In this
microscope, excitation of the fluorophore and detection of the fluorescence are done through the same
light path (i.e., through the objective).
b) Confocal microscope: In this type of fluorescence microscope, high‐resolution imaging of thick
specimens (without physical sectioning) can be analyzed using fluorescent-labeled dye.
c) Multiphoton microscope: In this type of microscope, multiphoton fluorescence excitation captures
high-resolution three-dimensional images of specimens tagged with highly specific fluorophores.
d) Total internal reflection fluorescence (TIRF) microscope: Total internal reflection fluorescence
microscopy (TIRFM) exploits the unique properties of an induced evanescent wave or field in a limited
specimen region immediately adjacent to the interface between two media having different refractive
indices.
Applications of Fluorescence Microscope:
• Fluorescence microscopy is widely used in diagnostic microbiology and microbial
ecology for enumerating bacteria in natural environments.
• It is used to identify structures in fixed and live biological samples.
• Fluorescence microscopy is a common tool for today’s life science research
because it allows the use of multicolor staining, labeling of structures within cells,
and the measurement of the physiological state of a cell.
• It is used to conduct viability studies on cell populations
• It is used to image the genetic material within a cell (DNA and RNA)
• It is used to view specific cells within a larger population with techniques such as
FISH
Electron Microscope (EM):
• Electron microscopy (EM) base on using an electron beam which is focused
into a small probe across the surface of a specimen.
• The first electromagnetic lens was developed in 1926 by Hans Busch.
• Electron microscope follows the same principle as compound microscope
but uses an electron beam as an illumination source instead of light.
• Electron microscopes allow biologists to explore cells in more details. To
observe the organelles such as : Mitochondria, Ribosomes, Endoplasmic
reticulum (ER), Golgi apparatus and Lysosomes.
• Heavy metals (such as lead) are used to stain cells before examination via
EM.
• The stain is more visible in organelles than in the surrounding cytoplasm.
Defects in a cell’s organelles are easily seen.
• Electron microscopes are used in science laboratories and many industries,
such as forensics, nanotechnology, and mining.
Scanning electron microscope (SEM)
• The mode of action for the SEM slimier to compound microscope however, an
electron beams behave like waves which focus via using a magnetic field rather
than uses of ordinary lenses.
• Metallic coating is required for the biological specimens. The electron
microscopes are used to achieve up to 100,000x magnification and more than 1000
x resolution than the light microscope.
• At these limits, sub-cellular structures can be easily observed.
• An electron microscope is a microscope that uses a beam of accelerated electrons
as a source of illumination.
• As the wavelength of an electron can be up to 100,000 times shorter than that of
visible light photons, electron microscopes have a higher resolving power than
light microscopes. They can reveal the structure of smaller objects.
Types:
Transmission electron microscope (TEM):
The original form of electron microscope, the transmission electron microscope
(TEM) uses a high voltage electron beam to illuminate the specimen and create an
image.
The electron beam is produced by an electron gun, commonly fitted with a tungsten
filament cathode as the electron source.
The electron beam is accelerated by an anode typically at +100 keV (40 to
40018keV) with respect to the cathode, focused by electrostatic and electromagnetic
lenses, and transmitted through the specimen that is in part transparent to electrons
and in part scatters them out of the beam.
When it emerges from the specimen, the electron beam carries information about the
structure of the specimen that is magnified by the objective lens system of the
microscope.
The spatial variation in this information (the "image") may be viewed by projecting
the magnified electron image onto a fluorescent viewing screen coated with a
phosphor or scintillator material such as zinc sulfide.
The advantages of electron diffraction over X-ray crystallography are that the
specimen need not be a single crystal or even a polycrystalline powder
The major disadvantage of the transmission electron microscope is the need for
extremely thin sections of the specimens, typically about 100 nanometers.
Biological specimens are typically required to be chemically fixed, dehydrated and
embedded in a polymer resin to stabilize them sufficiently to allow ultrathin
sectioning.
Sections of biological specimens, organic polymers and similar materials may
require special treatment with heavy atom labels in order to achieve the required
image contrast.
Scanning electron microscope (SEM):
The SEM produces images by probing the specimen with a focused electron beam that is
scanned across a rectangular area of the specimen (raster scanning).
When the electron beam interacts with the specimen, it loses energy by a variety of
mechanisms.
The lost energy is converted into alternative forms such as heat, emission of low-energy
secondary electrons and high-energy backscattered electrons, light emission
(cathodoluminescence) or X-ray emission, all of which provide signals carrying information
about the properties of the specimen surface, such as its topography and composition.
The image displayed by an SEM maps the varying intensity of any of these signals into the
image in a position corresponding to the position of the beam on the specimen when the
signal was generated.
Advantage of SEM is its variety called environmental scanning electron microscope
(ESEM) can produce images of sufficient quality and resolution with the samples being wet
or contained in low vacuum or gas. This greatly facilitates imaging biological samples that
are unstable in the high vacuum of conventional electron microscopes.