By M Mwale

Bsc CHN, Msc RH

 Objectives
At the end of the presentation learners should be able to:

 Explain the types of microscopes

 Explain resolution and magnification

 Explain techniques for the Observation of Objects

under Electron Microscopes
 State major groups of microorganisms and their
descriptions.

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 Microscope
 A microscope (Greek: mikron = small and scopeos =
to look).

 Microscope: Is an instrument for viewing objects that

are too small to be seen by the naked or unaided eye.

 Microscopy: The science of investigating small objects

using such an instrument is called microscopy

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 Microscope
 The microscope is an indispensible tool for examining the
structure cells and its components, because most cellular
structures are too small to be seen by the unaided eye.

 it is hundred times better at visualizing.

 Although light microscope and electron microscope differ

in many ways, they make use similar optical principles to
form images.

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 Types of microscopes
LIGHT MICROSCOPE :
 use sunlight or artificial light.
a. Bright field microscope.
b. Dark field microscope.
c. Phase contrast microscope.
d. Fluorescence microscope.
ELECTRON MICROSCOPE :
 use of electron.
a. Transmission electron microscope.
b. Scanning electron microscope.

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Types of microscopes ctd…
 Regardless of the type of microscope being used,
three elements are always needed to form an image:

 a source of illumination,

 a specimen to be examined, and

 a system of lenses that focuses the illumination on the

specimen and forms the image.

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 Light microscope
 In light microscope the source of illumination is visible
light and the lens system consists of series of glass lenses.
 The image can be either viewed directly through an eye
piece or focused on a detector, such as photographic film
or an electronic camera.
 In an electron microscope, the illumination source is a
beam of electrons emitted by a heated tungsten filament,
and the lens system consists of a series of electromagnets.

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 Bright field microscope
 In BFM, the microscopic field (the area observed) is highly
lightened and the microorganisms appear dark because they
absorb some of the light.

 Produces a dark image against a brighter background.

 Staining the specimen with dye greatly increases their light

absorbing ability, resulting in greater contrast and color
differentiation.

 2 types: Simple & Compound

 Simple - Contain a single magnifying lens.


7/26/2024 Compound BIOL
- Series
110 2019lenses for magnification.
of 9
 Instrumentation

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 Dark field microscopy
 The effect produced by the dark field technique is
that of a dark back ground against which objects are
brilliantly illuminated.

 This is accomplished by equipping the light

microscope with special kind of condenser that
transmits a hallow cone of light from the source of
illumination.
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 Phase contrast microscope
 Phase contrast microscope is useful for observing cells
that are intact and living.

 To understand the basis of phase contrast microscope

we must first recognize that a beam of light is made
up of many individual rays of light.

 As the rays pass from the light source through the

specimen, their velocity may be affected by the
physical properties of the specimen.
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 Optics of the Phase contrast
microscope

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Phase contrast microscope ctd…
 Usually the velocity of the rays is slow down, to
varying extents by different regions of the specimen,
resulting in a change in phase relative to light waves
that have not passed through the specimen ( light
waves are said to traveling in phase when crest and
trough of the waves match each other)

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 Differential interference contrast
microscope
 Is similar to phase contrast but it is more sensitive
because it employs a special prism to split the light
beam into two separate rays.

 The image appears in 3D due to shadow casting

illusion that arises because differences in phase are
positive on one side of the cell but negative on the
opposite of the cell.
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 Optics of the Differential
interference contrast microscope

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 Fluorescence microscopy
 Useful in observing specific molecules located within
the cells.

 Fluorescence refers to absorbing light by a molecule

and ends with emission of light with a longer wave
length and lesser energy.

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 Electron microscope
 Electron microscopes are of two basic designs:

1. Transmission electron microscope

2. Scanning electron microscope

 TEM and SEM are similar in that each employs bean

of electrons to produce an image.

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 Electron microscopes – use a beam of electrons
instead of a beam of light to magnify the image

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 Transmission electron
microscope
 It is so named because it forms an image from
electrons that are transmitted through the specimen
being examined.

 Instead of light source they use electron source which

is a tungsten filament in this case, (cathode)
electromagnets are used instead of lens.

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 Transmission electron
microscope

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 Scanning electron microscope
 SEM produces images from electrons deflected from a
specimen’s outer surface ( rather than electrons
transmitted through the specimen).
 It generates images by scanning the specimen’s surface
with a beam of electrons.
 The accelerating voltage is low( 5-30KV) compared
to TEM, the vacuum system and electron source are
similar

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Scanning electron microscope

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 Basic principle of microscopy
 The wavelength of the illumination source sets a limit on
how small an object can be seen( both light and electron
microscope behave as waves).
 When a beam of light(or electron) encounters a specimen,
the specimen alters the physical characteristics of the
illuminating beam
 And because an object can be detected only by its effect
on the wave, the wavelength must be comparable in size
to the object that is to be detected.
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  The wavelength of electrons are very much similar
shorter than those of photon.
 When waves of light or electrons pass through a lens
and are focused, the image that is formed results from
a property of waves called Inference
 the process by which two or more waves combine to
reinforce or cancel one another, producing a wave
equal to the sum of the two combining waves

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  Thus, the image that you see when you look at a specimen
through a series of lenses is really just a pattern of either
additive or canceling interference of wave that went through
the lenses, a phenomenon known as diffraction.
 In a light microscope, glass lenses are used to direct photons,
where as an electron microscope uses electron magnets as
lenses to direct electrons
 Yet both kinds of lenses have two fundamental properties in
common: focal length and Angular aperture (Numerical
aperture, NA).

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  The focal length is the distance between the midline of the
lens and the point at which rays passing through the lens
converge to a focus.
 The angular aperture is a measure of how much of the
illumination that leaves the specimen actually passes
through the lens or it’s a measure of light gathering ability
by the lens, an innate property of objective lens.
 Angular aperture of a lens is one of the factors influencing
a microscope’s resolution

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 Focal length of a lens

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 Low -aperture & High -aperture lens

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 What does a Microscope do?
 A Microscope has 2 jobs

1. Magnification to make something appear larger

2. Resolution the ability to distinguish two objects as

being separate from each other

 Resolution, not magnification, increases the amount

of detail you can see in an object!

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 Resolution
 Resolution is defined as the minimum distance that can separate
two points that still remain identifiable as separate points when
viewed through the microscope.
 is the shortest distance between two points that a user can still
see as separate images under the microscope.
 Resolution is governed by 3 factors:

1. the wave length of the light used to illuminate the specimen,

2. the angular aperture,
3. the refractive index of the medium surrounding the specimen.

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  Refractive index is a measure of the change in the
velocity of the light as it passes from one medium to
another.

 Limit of resolution: the limit of resolution is the

smallest distance by which two objects can be
separated and still be distinguishable as two separate
objects.
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 Magnification
 Degree of enlargement.

 No of times the length, breadth or diameter, of an object is

multiplied.

 It depends upon - Optical tube length

- Focal length of objective

- Magnifying power of eye piece

 TOTAL MAGNIFICATION:

magnification of the eyepiece x magnification of the objective.

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  Magnification beyond the resolving power is of no
value since the larger image will be less distinct in
detail and fuzzy in appearance.

 Laboratory microscopes are equipped with three

objectives each capable of a different degree of
magnification; Oil immersion, high dry and low
power objective.

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 Sample preparation
 In each case, the methods is complicated, time
consuming and costly compared with methods used
for light microscopy.

 More over living cells cannot be examined because

the electron microscope requires specimen to be
subjected to a vacuum.

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 Techniques for the Observation of
Objects under Electron Microscopes
 Ultra thin sectioning and staining are common preparation techniques
for Transmission Electron Microscope

 Ultra thin sections not more than 5-10nm are fixed and stained
initially by osmium tetroxide to improve in contrast of structures

Ultra-thin Sectioning: An intact microbial cell is too thick to allow

visualization of its internal fine structures under electron microscope.

 For observation of the intracellular fine structures, the cell is

embedded in a block of plastic material and cut into ultra-thin
sections

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 Techniques for the Observation of Objects under
Electron Microscopes ctd…
 Cryofixation - high pressure freezing is one such way

fixing specimen efficiently preparing for the specimen for

sectioning.

 Using ultra microtome the embedded specimen is sliced

into thin section, a glass or diamond knife is used.

 This is again stained with uranium and lead which shall

give greater electron density to specific parts of the cells.

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 Techniques for the Observation of Objects under
Electron Microscopes ctd…
 Negative staining: which is one of the simplest techniques in
transmission electron microscopy, intact specimens are simply
visualized in relief against a darkly stained background.

 This technique is followed to observe minute details of very

small objects, such as viruses and bacteria flagella

 Stains used are uranyl acetate/phosphotungstic acid.

 Used for examining shape and surface of very small objects like
virus, isolated organelles.

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Techniques for the Observation of Objects under
Electron Microscopes ctd…
 Shadowing technique: spraying a thin layer of an electron dense
metal such as gold/platinum at angle across the surface of a specimen
using the metal replica the structure is examined.
 Provides information as to the shape of the object

 Freeze fracturing: samples are frozen at such low temperature are too
hard to be cut (liquid nitrogen/liquid helium).
 Instead they fracture along lines of natural weakness-the hydrophobic
interior of membrane, in most cases.
 Platinum /carbon shadowing is then used to create a replica of the
fracture surface.

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 Techniques for the Observation of Objects under
Electron Microscopes ctd…
 Specimen for Scanning Electron Microscope: involves
fixation but not sectioning, tissue is fixed in aldehyde, post
fixed is osmium tetraoxide and dehydrated by processing.
 Then tissue is placed in liquid CO2 in a heavy metal
canister called a critical point dryer, which is used to dry
the specimen under condition of controlled temperature
and pressure.
 Dried specimen is coated with gold /palladium and ready
to examine.

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 Microorganisms
 Microorganisms, as their name implies, are so small that they
usually require a microscope to see.

 The vast majority of microbes on the earth pose no real threat

to humans, plants or animals

 They aid in decomposition, decay and helps in digesting food.

 However, there are some microorganisms which negatively

impact our lives, causing illness, bad odours and damaging
products and surfaces.

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 Nature of Microorganisms
 Small number of microorganisms called pathogens

 cause disease- which negatively impact our lives, causing illness, bad
odours and damaging products and surfaces.

 Most are harmless, called non-pathogens

 aid decomposition, decay and even helps in food digestion.

 Some may be pathogenic in one situation, and not in others (e.g.,

Escherichia coli [E. coli] bacterium), certain varieties cause diarrhoea

 Normally in intestinal tract, breaks down waste, aids in synthesis of Vit K

(helps control bleeding)

 If E. coli moves to another part of body, major problems result

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 Major groups of microorganisms and their
descriptions.
Bacteria
 Most well-known microorganism that infect humans- makes a large
group of microorganisms

 are a member of the prokaryotes; they have no nucleus within the

cell and contain no organelles.

 Within bacteria there are two classes,

1. Gram positive bacteria which have thicker cell wall

2. Gram negatives which have a thinner layer sandwiched between

an inner and outer membrane.

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 Major groups ctd…
 One-celled plants classified by size, shape and arrangement

 Arrangement is way to identify exact species

 Some are

 Round cocci

 Streptococci chains

 Staphylococci in groups or clusters

 Diplococci in pairs

 Micrococci

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 Major groups ctd…
 Bacteria are the only microorganisms which can live
harmlessly within the human body, often aiding
bodily functions such as digestion.

 However of all the ‘living’ microorganisms, Bacteria

cause the most problems in terms of disease in
humans, despite only relatively few bacteria being
dangerous.

 Grows best at 98.6 F in moist, dark environment

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 Fungi
 are eukaryotes which means they have a defined nucleus and
organelles.

 The cells are larger than prokaryotes such as bacteria.

 Fungal colonies can be visible to the human eye once they have
achieved a certain level of growth, for example mould on bread.

 Fungi can be split into three main groups,

1. Moulds which display thread-like (filamentous) growth and

multicellular structures,

2. Yeasts which are typically non-filamentous and can be single celled

3. Mushrooms which possess a fruiting body for production of spores.

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 Major groups ctd…
 Fungi can be problematic for the
immunocompromised and contain significant
pathogens which can cause disease in plants.
 Responsible for conditions such as ringworm,
athletes foot, and yeast infections
 This group is known for widespread use in the food
industry, for production of beer and other foodstuffs.

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 Major groups ctd…
Viruses
 are the smallest of all microorganisms and difficult to destroy -
Only be seen through electron microscope
 They essentially consist of nucleic acid (DNA or RNA,) and a
protein coat.
 A virion (a virus particle) requires a host cell in order to
replicate. (Grow and reproduce only inside other living cells)
 All viruses infect cells and hijack the host cellular machinery to
make copies of its own genetic material in a process called
replication

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 Major groups ctd…
 Humans are infected with viruses by contact with other living
beings and it is spread by contact with blood and body fluids

 Diseases associated with viruses are common cold, chickenpox,

herpes, hepatitis B, and HIV/AIDS

 Health care workers are at great risk of being exposed to blood

and body fluids

 It is important to use PPEs all the time one is in contact with

body fluids.

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 Major groups ctd…
 Algae
 are a more difficult to define group of organisms, containing
both prokaryotes and eukaryotes by some definitions.
 Unlike other microorganisms algae are typically photo
synthesisers and are typically found in marine environments.
 Prokaryotic algae, or Cyanobacteria, are often called blue-green
algae although some definition or opinions will state that algae
are eukaryotes only (that they are essentially small aquatic
plants).

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 Major groups ctd…
Protozoa
 Consist of a large group of one-cell organisms that do not

have a rigid cell wall and are found in contaminated

water supplies, marine habitat and moist soil.
 A small number are responsible for intestinal infections of

humans and others invade the blood, lungs, liver, or brain

 cause diseases such as malaria, trichomoniasis, and

amoebic dysentery
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 Major groups ctd…
Archaea
 Archaea are unicellular organisms that make up the third
domain of organisms on earth
 Like bacteria, however, archaea are prokaryotes that share
certain characteristics with bacteria (this is one of the reasons
archaea were previously thought to be a type of bacteria).
 Due to their ability to survive extreme conditions, they can be
found in a variety of environments ranging from lakes and soil
to the Dead Sea and the deepest parts of the ocean (ocean
floor).
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 References
 http//www.life.umd.edu/classroom/bsci424/Pathogen
Descriptions/StreptococcusImages.htm

 https://www.biocote.com/blog/microorganisms-an-
introduction/ 24/10/19

 https://sciencing.com/function-microscope-6575328.html

 https://www.microscopemaster.com/unicellular-
organisms.html

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