Dr.

Bharti Wadekar

History of Microscope
 The evolution of the Microbiology field put to perspective the need to identify, view,
observe and understand microorganisms, including their structural morphologies and
mechanisms. Microbiology’s scope is to study organisms and minute agents that can
only be examined and observed with a microscope.
 Although scientifically, the first simple microscope was discovered by two Dutch
scientists, Zaccharias Janssen and his father, Hans who made spectacles, were the first
to experiment with their lenses by combining lenses in a tube and observed that the
objects that were nearby, appeared closer and larger. Despite not being included as a
scientific discovery, this act paved the way for scientific evolution.
 From the History of Microbiology, Antony Van Leeuwenhoek an amateur
Microbiologist made the first simple microscope, that enabled him to observe the
presence of tiny living organisms in pond water that appeared like dots. His simple
microscope was made up of a double convex glass lens that was held between two silver
plates.
 The application of microscopy in Microbiology enhanced the visualization of cells and
microorganisms by magnifying their images to make them larger.

The light microscope is also known as an optical microscope.

 1. Simple Microscope- Definition, Principle,
Parts, Applications
 The optical microscope often referred to as the light microscope, is a type of microscope
that uses visible light and a system of lenses to magnify images of small subjects.
 There are two basic types of optical microscopes:
1. Simple microscopes
2. Compound microscopes.

Simple Microscope Definition

 A simple microscope is one that uses a single lens for magnification, such as a
magnifying glass while a compound microscope uses several lenses to enhance the
magnification of an object.
 It uses a lens to enlarge an object through angular magnification alone, giving the
viewer an erect enlarged virtual image.
 The use of a single convex lens or groups of lenses is found in simple magnification
devices such as the magnifying glass, loupes, and eyepieces for telescopes and
microscopes.
 It is actually a convex lens of small focal length, which is used for seeing the magnified
images of small objects.
Principle of Simple Microscope

A simple microscope works on the principle that when a tiny object is placed within its focus,
a virtual, erect and magnified image of the object is formed at the least distance of distinct
vision from the eye held close to the lens.

Magnification of Simple Microscope

The magnifying power of a simple microscope is given by:
M = 1 + D/F
Where D = the least distance of distinct vision
F = focal length of the convex lens
 The focal length of the convex lens should be small because smaller the focal length of
the lens, greater will be its magnifying power.
 The maximum magnification of a simple microscope is about 10, which means that the
object will appear 10 times larger by using the simple microscope of maximum
magnification.

Instrumentation of Simple Microscope

 Figure: Simple Microscope Diagram

 The parts of a simple microscope maybe:

 (i) Mechanical parts
 (ii) Optical parts

Mechanical parts:
These parts support the optical parts and help in their adjustment for focusing the object.
They include the following components:
1. Metal Stand:
 It has a heavy base plate and a vertical rod fitted to it, which provide support and
stability to other parts of the microscope.
2. Stage:
 It is a rectangular metal plate fitted to the vertical rod.
 It has a central hole for light to pass from below.
 Slide with the specimen to be observed is kept on the stage, in such a way that, the
specimen remains just on the central hole.
 Some microscopes have a pair of slanting wings projecting from both sides of the stage.
They provide support to hand for manipulating the object.

Optical parts:
These parts are involved in passing the light through the object (specimen) and magnifying its
size.
The components of the optical parts are as follows:
1. Mirror:
  A plano-convex mirror is fitted below the stage to the vertical rod by means of a frame.
 It focuses the surrounding light on the object to be observed.
2. Lens:
 A biconvex lens is fitted above the stage, to the vertical rod, by means of a frame.
 It magnifies the size of the object and the enlarged virtual image formed is observed by
keeping the eye above it.
 For proper focusing, the lens can be moved up and down by the frame.

Applications
 It is usually used for the study of microscopic algae, fungi, and biological specimens.
 It is commonly used by watchmakers to see the magnified view of small parts of a
watch.
 It is also used by the jewelers to see the magnified view of the fine parts of jewelry.
 It is used to see the enlarged image of letters of a book, textures of fibers or threads of
a cloth.
 It is used to see the magnified view of different particles of different types of soils.
 It is used by palmists to see an enlarged view of the lines of our hand.
 It is used by skin specialists to find out various diseases of the skin.
 It is also used to see the details of stamp and engravings.

2. Compound microscope- definition, labeled

diagram, parts, uses

Compound Microscope Definition

 The term microscope can be split into two separate words, ‘micro’ and ‘scope’, where
the term ‘micro’ means small or tiny, and ‘scope’ means to view or to observe.
Therefore, a microscope can be understood as an instrument to observe tiny elements.
 The optical microscope often referred to as the light microscope, is a type of microscope
that uses visible light and a system of lenses to magnify images of small subjects.
 There are two basic types of optical microscopes:
1. Simple microscopes
2. Compound microscopes
 The term “compound” in compound microscopes refers to the microscope having more
than one lens.
 Devised with a system of combination of lenses, a compound microscope consists of
two optical parts, namely the objective lens and the ocular lens.
Working Principle of the Compound Microscope
Compound microscopes have a combination of lenses that enhances both magnifying powers
as well as the resolving power.
 The specimen or object, to be examined is usually mounted on a transparent glass slide
and positioned on the specimen stage between the condenser lens and objective lens.
 A beam of visible light from the base is focused by a condenser lens onto the specimen.
 The objective lens picks up the light transmitted by the specimen and creates a
magnified image of the specimen called the primary image inside the body tube. This
image is again magnified by the ocular lens or eyepiece.
 When higher magnification is required, the nose piece is rotated after low power
focusing to bring the objective of a higher power (generally 45X) in line with the
illuminated part of the slide.
 Occasionally very high magnification it required (e.g. for observing bacterial cell). In
that case, an oil immersion objective lens (usually 100X) is employed.
 The common light microscope is also called a bright-field microscope because the
image is produced amidst a brightly illuminated field. The image appears darker
because the specimen or object is denser and somewhat opaque than the surroundings.
Part of the light passing through or object is absorbed.

Magnification of compound microscope

In order to ascertain the total magnification when viewing an image with a compound light
microscope, take the power of the objective lens which is at 4x, 10x or 40x and multiply it by
the power of the eyepiece which is typically 10x.
Therefore, a 10x eyepiece used with a 40X objective lens will produce a magnification of 400X.
The naked eye can now view the specimen at magnification 400 times greater and so
microscopic details are revealed.
Alternatively, the magnification of the compound microscope is given by:
m = D/ fo * L/fe
where, D =Least distance of distinct vision (25 cm)
L = Length of the microscope tube
fo = Focal length of the objective lens
fe = Focal length of the eye-piece lens

Parts of a Compound Microscope

Eyepiece And Body Tube.
 The eyepiece is the lens through which the viewer looks to see the specimen.
 It usually contains a 10X or 15X power lens.
 The body tube connects the eyepiece to the objective lenses.
Objectives and Stage Clips
 Objective Lenses are one of the most important parts of a Compound Microscope.
 They are the closest to the specimen.
 A standard Microscope has three to four Objective Lenses which range from 4X to
100X.
 Stage Clips are metal clips that held the slide in a place.
Arm and Base
 The Arm connects the Body Tube to the base of the Microscope.
 The Base supports the Microscope and its where Illuminator.
Illuminator and Stage
 The illuminator is the light source for a microscope.
 A compound light microscope mostly uses a low voltage bulb as an illuminator.
 The stage is the flat platform where the slide is placed.
Nosepiece and Aperture
 Nosepiece is a rotating turret that holds the objective lenses.
 The viewer spins the nosepiece to select different objective lenses.
 The aperture is the middle of the stage that allows light from the illuminator to reach
the specimen.
Condenser, Iris diaphragm, and Diaphragm
 A condenser gathers and focuses light from the illuminator onto the specimen being
viewed.
 Iris diaphragm adjusts the amount of light that reaches the specimen.
 The diaphragm is a five holed disk placed under the stage.
 Each hole is of a different diameter. By turning it, you can vary the amount of light
passing through the stage opening.

Applications
 A compound microscope is of great use in pathology labs so as to identify diseases.
  Various crime cases are detected and solved by drawing out human cells and examining
them under the microscope in forensic laboratories.
 The presence or absence of minerals and the presence of metals can be identified using
compound microscopes.
 Students in schools and colleges are benefited by the use of a microscope for conducting
their academic experiments.
 It helps to see and understand the microbial world of bacteria and viruses, which is
otherwise invisible to the naked eye.
 Plant cells are examined and the microorganisms thriving on it can be ascertained with
the help of a compound microscope. Thereby, a compound microscope has proved to
be crucial to biologists.

Advantages
 Simplicity and its convenience.
 A compound light microscope is relatively small, therefore it’s easy to use and simple
to store, and it comes with its own light source.
 Because of their multiple lenses, compound light microscopes are able to reveal a great
amount of detail in samples.

3. Brightfield Microscope (Compound Light

Microscope)

Brightfield Microscope Definition

Brightfield Microscope is also known as the Compound Light Microscope. It is an
optical microscope that uses light rays to produce a dark image against a bright background. It
is the standard microscope that is used in Biology, Cellular Biology, and Microbiological
Laboratory studies.
This microscope is used to view fixed and live specimens, that have been stained with basic
stains which gives a contrast between the image and the image background. It is specially
designed with magnifying glasses known as lenses that modify the specimen to produce an
image seen through the eyepiece.

Principle of Brightfield Microscope

For a specimen to be the focus and produce an image under the Brightfield Microscope, the
specimen must pass through a uniform beam of the illuminating light. Through differential
absorption and differential refraction, the microscope will produce a contrasting image.
The specimens used are prepared initially by staining to introduce color for easy contracting
characterization. The colored specimens will have a refractive index that will differentiate it
from the surrounding, presenting a combination of absorption and refractive contrast.
The functioning of the microscope is based on its ability to produce a high-resolution image
from an adequately provided light source, focused on the image, producing a high-quality
image.
The specimen which is placed on a microscopic slide is viewed under oil immersion or/and
covered with a coverslip.

Parts of Brightfield Microscope

The brightfield microscope is made up of various parts, including

 Eyepiece (Ocular lens) – it has two eyepiece lenses at the top of the microscope which
focuses the image from the objective lenses. this is where you see the formed image
from, with your eyes.
 The objective lenses which are made up of six or more glass lenses, which make a
clear image clear from the specimen or the object that is being focused.
 Two focusing knobs i.e the fine adjustment knob and the coarse adjustment knob,
found on the microscopes’ arm, which can move the stage or the nosepiece to focus on
the image. Their function is to ensure the production of a sharp image with clarity.
 The stage is found just below the objectives and this is where the specimen is placed,
allowing movement of the specimen around for better viewing with the flexible knobs
and it is where the light is focused on.
 The condenser: It is mounted below the stage which focuses a beam of light onto the
specimen. It can be fixed or movable, to adjust the quality of light, but this entirely
depends on the microscope.
 The arm: This is a sturdy metallic backbone of the microscope, used to carry and move
the microscope from one place to another. They also hold the microscope base which
is the stand of the microscope. The arm and the base hold all the microscopic parts.
 It has a light illuminator or a mirror found at the base or on the microscope’s
nosepiece.
 The nosepiece has about two to five objective lenses with different magnifying power.
It can move round to any position depending on the objective lens to focus on the image.
 An aperture diaphragm (contrast): It controls the diameter of the beam of light that
passes through the condenser. When the condenser is almost closed, the light comes
through to the center of the condenser creating high contrast and when the condenser is
widely open, the image is very bright with very low contrast.
Magnification by Brightfield Microscope
 The objective lenses are the main lenses used for focusing the image, on the condenser.
This produces an enlarged clear image that is then magnified again by the eyepiece to
form the primary image that is seen by the eyes.
 During imaging, the objective lenses remain parfocal in that, even when the objective
lens has changed the image still remains focused. The image seen at the eyepiece is the
enlarged clear image of the specimen, known as the virtual image.
 The magnification of the image is determined by the magnification of the objective
against the magnification of the eyepiece lens. The objectives have a magnification
power of 40x-1000x depending on the type of brightfield microscope while the
eyepiece lens has a standard magnification power of 10x.
 Therefore to calculate:
Total Magnification power = Magnification of the objective lens x Magnification of the
eyepiece
 For example: if the magnification of the objective is 45x and that of the eyepiece is 10x,
the total magnification of the specimen will be 450x.
 The magnification is standard, i.e not too high nor too low, and therefore depending on
the magnification power of the lenses, it will range between 40X and 100oX.
 The objective lens enlarges the image which can be viewed, a characteristic known as
resolution. Resolution according to Prescott, is the ability of a lens to separate or
distinguish between small objects closely linked together.
 Whereas the eyepiece magnifies the image at the end of the viewing, its magnification
range is lower than that of the objective lens at 8X-12X (10X standard) and that of the
objective lens at 40X-100X, magnification, and resolution of the microscope is highly
dependant on the objective lens.
Applications of Brightfield microscope
Brightfield Microscope is used in several fields, from basic biology to understanding cell
structures in cell Biology, Microbiology, Bacteriology to visualizing parasitic organisms in
Parasitology.
Most of the specimens to viewed are stained using special staining to enable visualization.
Some of the staining techniques used include Negative staining and Gram staining.
Some of its applications include:
1. Used to visualize and study the animal cells
2. Used to visualize and study plant cells.
3. Used to visualize and study the morphologies of bacterial cells
4. Used to identify parasitic protozoans such as Paramecium.
Advantages of Brightfield Microscope
1. It is simple to use with few adjustments involved while viewing the image.
2. It can be used to view both stained and unstained.
3. The optics of the microscope do not alter the color of the specimen.
4. The microscope can be adjusted and modified for better viewing such as installing a
camera, to form a digital microscope or in the way image illumination is done such as
by use of fluorochromes on the specimen and viewing under a dark environment,
forming a darkfield microscope.
Disadvantages
1. The aperture diaphragm may cause great contrast which may distort the outcome of the
image, therefore iris diaphragm is preferred.
2. It can not be used to view live specimens such as bacterial cells. Only fixed specimens
can be viewed under the brightfield microscope.
3. Maximum magnification of the brightfield microscope is 100x but modification can
readjust the magnification to 1000x which is the optimum magnification of bacterial
cells.
4. It has low contrast hence most specimens must be stained for them to be visualized.
5. Use of oil immersion may distort the image
6. The use of coverslip may damage the specimen
7. Staining may introduce extraneously unwanted details into the specimen or contaminate
the specimen.
8. It is tedious to stain the specimen before visualizing it under the brightfield microscope.
9. The microscope needs a strong light source for magnification and sometimes the light
source may produce a lot of heat which may damage or kill the specimen.
 [Link] Microscope- Definition,
Principle and Uses
Darkfield Microscope Definition
 Microbiology, the branch of science that has so vastly extended and expanded our
knowledge of the living world, owes its existence to Antoni van Leeuwenhoek.
 In 1673, with the aid of a crude microscope consisting of a biconcave lens enclosed in
two metal plates, Leeuwenhoek introduced the world to the existence of microbial
forms of life.
 Over the years, microscopes have evolved from the simple, single-lens instrument of
Leeuwenhoek, with a magnification of 300 X, to the present-day electron microscopes
capable of magnifications greater than 250,000X.
 Microscopes are designated as either light microscopes or electron microscopes.
 Light microscopes use visible light or ultraviolet rays to illuminate specimens. They
include brightfield, darkfield, phase-contrast, and fluorescent instruments.
 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.

Principle of the Darkfield Microscope

 A dark field microscope is arranged so that the light source is blocked off, causing light
to scatter as it hits the specimen.
 This is ideal for making objects with refractive values similar to the background appear
bright against a dark background.
 When light hits an object, rays are scattered in all azimuths or directions. The design of
the dark field microscope is such that it removes the dispersed light, or zeroth order, so
that only the scattered beams hit the sample.
 The introduction of a condenser and/or stop below the stage ensures that these light rays
will hit the specimen at different angles, rather than as a direct light source above/below
the object.
 The result is a “cone of light” where rays are diffracted, reflected and/or refracted off
the object, ultimately, allowing the individual to view a specimen in dark field.

This is to say:
1. The dark-ground microscopy makes use of the dark-ground microscope, a special type
of compound light microscope.
2. The dark-field condenser with a central circular stop, which illuminates the object with
a cone of light, is the most essential part of the dark-ground microscope.
3. This microscope uses reflected light instead of transmitted light used in the ordinary
light microscope.
4. It prevents light from falling directly on the objective lens.
5. Light rays falling on the object are reflected or scattered onto the objective lens with
the result that the microorganisms appear brightly stained against a dark background.
Uses of Darkfield Microscope
The dark ground microscopy has the following uses:
 It is useful for the demonstration of very thin bacteria not visible under ordinary
illumination since the reflection of the light makes them appear larger.
 This is a frequently used method for rapid demonstration of Treponema pallidum in
clinical specimens.
 It is also useful for the demonstration of the motility of flagellated bacteria and
protozoa.
 Darkfield is used to study marine organisms such as algae, plankton, diatoms, insects,
fibers, hairs, yeast and protozoa as well as some minerals and crystals, thin polymers
and some ceramics.
 Darkfield is used to study mounted cells and tissues.
 It is more useful in examining external details, such as outlines, edges, grain boundaries
and surface defects than internal structure.

Advantages of Darkfield Microscope

 Dark-field microscopy is a very simple yet effective technique.
 It is well suited for uses involving live and unstained biological samples, such as a
smear from a tissue culture or individual, water-borne, single-celled organisms.
 Considering the simplicity of the setup, the quality of images obtained from this
technique is impressive.
 Dark-field microscopy techniques are almost entirely free of artifacts, due to the nature
of the process.
 A researcher can achieve a dark field by making modifications to his/her microscope
.

Limitations of Darkfield Microscope

 The main limitation of dark-field microscopy is the low light levels seen in the final
image.
 The sample must be very strongly illuminated, which can cause damage to the sample.
[Link] contrast Microscopy- definition,
principle, parts, uses

Phase contrast microscopy definition

 Unstained living cells absorb practically no light. Poor light absorption results in
extremely small differences in the intensity distribution in the image. This makes the
cells barely, or not at all, visible in a brightfield microscope. 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.
 It was first described in 1934 by Dutch physicist Frits Zernike.

Principle of Phase contrast Microscopy

When light passes through cells, small phase shifts occur, which are invisible to the human
eye. In a phase-contrast microscope, these phase shifts are converted into changes in amplitude,
which can be observed as differences in image contrast.

The Working of Phase contrast Microscopy

1. Partially coherent illumination produced by the tungsten-halogen lamp is directed
through a collector lens and focused on a specialized annulus (labeled condenser
annulus) positioned in the substage condenser front focal plane.
2. Wavefronts passing through the annulus illuminate the specimen and either pass
through undeviated or are diffracted and retarded in phase by structures and phase
gradients present in the specimen.
3. Undeviated and diffracted light collected by the objective is segregated at the rear focal
plane by a phase plate and focused at the intermediate image plane to form the final
phase-contrast image observed in the eyepieces.

Parts of Phase contrast Microscopy

Phase-contrast microscopy is basically a specially designed light microscope with all the basic
parts in addition to which an annular phase plate and annular diaphragm are fitted.
The annular diaphragm
 It is situated below the condenser.
 It is made up of a circular disc having a circular annular groove.
 The light rays are allowed to pass through the annular groove.
 Through the annular groove of the annular diaphragm, the light rays fall on the
specimen or object to be studied.
 At the back focal plane of the objective develops an image.
 The annular phase plate is placed at this back focal plane.
The phase plate
 It is either a negative phase plate having a thick circular area or a positive phase plate
having a thin circular groove.
 This thick or thin area in the phase plate is called the conjugate area.
 The phase plate is a transparent disc.
 With the help of the annular diaphragm and the phase plate, the phase contrast is
obtained in this microscope.
 This is obtained by separating the direct rays from the diffracted rays.
 The direct light rays pass through the annular groove whereas the diffracted light rays
pass through the region outside the groove.
 Depending upon the different refractive indices of different cell components, the object
to be studied shows a different degree of contrast in this microscope.
Applications of Phase contrast Microscopy
To produce high-contrast images of transparent specimens, such as
1. living cells (usually in culture),
2. microorganisms,
3. thin tissue slices,
4. lithographic patterns,
5. fibers,
6. latex dispersions,
7. glass fragments, and
8. subcellular particles (including nuclei and other organelles).
Applications of phase-contrast microscopy in biological research are numerous.

Advantages
 Living cells can be observed in their natural state without previous fixation or labeling.
 It makes a highly transparent object more visible.
 No special preparation of fixation or staining etc. is needed to study an object under a
phase-contrast microscope which saves a lot of time.
 Examining intracellular components of living cells at relatively high resolution. eg: The
dynamic motility of mitochondria, mitotic chromosomes & vacuoles.
 It made it possible for biologists to study living cells and how they proliferate through
cell division.
 Phase-contrast optical components can be added to virtually any brightfield
microscope, provided the specialized phase objectives conform to the tube length
parameters, and the condenser will accept an annular phase ring of the correct size.

Limitations
 Phase-contrast condensers and objective lenses add considerable cost to a microscope,
and so phase contrast is often not used in teaching labs except perhaps in classes in the
health professions.
 To use phase-contrast the light path must be aligned.
 Generally, more light is needed for phase contrast than for corresponding bright-field
viewing, since the technique is based on the diminishment of the brightness of most
objects.
OVERVIEW OF ALL MICROSCOPE
What is a light microscope?

 A light microscope is a biology laboratory instrument or tool, that uses visible light to
detect and magnify very small objects, and enlarging them.
 They use lenses to focus light on the specimen, magnifying it thus producing an image.
The specimen is normally placed close to the microscopic lens.
 Microscopic magnification varies greatly depending on the types and number of lenses
that make up the microscope. Depending on the number of lenses, there are two types
of microscopes i. e Simple light microscope (it has low magnification because it uses a
single lens) and the Compound light microscope (it has a higher magnification
compared to the simple microscope because it uses at least two sets of lenses, an
objective lens, and an eyepiece). The lenses are aligned in that, they can be able to bend
light for efficient magnification of the image.
 The functioning of the light microscope is based on its ability to focus a beam of light
through a specimen, which is very small and transparent, to produce an image. The
image is then passed through one or two lenses for magnification for viewing. The
transparency of the specimen allows easy and quick penetration of light. Specimens can
vary from bacterial to cells and other microbial particles.
Light Microscope- definition, principle, types,
parts, magnification
As mentioned earlier, light microscopes visualize an image by using a glass lens and
magnification is determined by, the lens’s ability to bend light and focus it on the specimen,
which forms an image. When a ray of light passes through one medium into another, the ray
bends at the interface causing refraction. The bending of light is determined by the refractive
index, which is a measure of how great a substance slows the speed of light. The direction and
magnitude of the bending of the light are determined by the refractive indexes of the two
mediums that form the interface.
A medium with a lower refractive index such as glass to air, it normally speeds up the light
penetration and making light bend away from the normal and when light is passed through a
medium with a greater refractive index such as air to glass, it normally slows down and bends
towards the normal, perpendicularly to the surface.
If an object is put between these two mediums i.e between water and air, in this case, a prism,
the prism will bend the light at an angle. This is how the microscopic lenses work, they bend
the light at an angle. The lens (convex) on receiving the light rays, it focuses the rays at a
specific point known as the focal point (F-point). The measure of distance from the center of
the lens and the focal point is known as the focal length.
A microscope uses lenses whose strength is predetermined, in that, the strength of a lens is
directly related to the focal length i.e short focal length magnifies objects more than lenses with
a long focal length.
Microscopy works strictly with a factor of resolution whereby resolution being the ability of a
lens to be able to differentiate small objects that are closely packed together. The resolution of
a light microscope is determined by a numerical aperture of its lens system and by the
wavelength of the light it employs; a numerical aperture a definition of the light wavelengths
produced when the specimen is illuminated.
A minimum distance (d) between two objects that distinguishes then to be two separate entities,
determined by the wavelengths of the light can be calculated by an Abbe equation using the
wavelength of the light that illuminated the specimen (Lambda, λ) and the numerical aperture
(NA, n sin Ɵ) i.e. d=0.5 λ/n sin Ɵ

Types of light microscopes (optical microscope)

With the evolved field of Microbiology, the microscopes
used to view specimens are both simple and compound light microscopes, all using lenses. The
difference is simple light microscopes use a single lens for magnification while compound
lenses use two or more lenses for magnifications. This means, that a series of lenses are placed
in an order such that, one lens magnifies the image further than the initial lens.
The modern types of Light Microscopes include:
1. Bright field Light Microscope
2. Phase Contrast Light Microscope
3. Dark-Field Light Microscope
4. Fluorescence Light Microscope
Brightfield Light Microscope (Compound light
microscope)
 This is the most basic optical Microscope used in microbiology laboratories which
produces a dark image against a bright background. Made up of two lenses, it is
widely used to view plant and animal cell organelles including some parasites such
as Paramecium after staining with basic stains.
 Its functionality is based on being able to provide a high-resolution image, which highly
depends on the proper use of the microscope. This means that an adequate amount of
light will enable sufficient focusing of the image, to produce a quality image.
 It is also known as a compound light microscope.
Parts of a bright-field microscope (Compound light microscope)

It is composed of:
 Two lenses which include the objective lens and the eyepiece or ocular lens.
 Objective lens is made up of six or more glasses, which make the image clear from the
object
 The condenser is mounted below the stage which focuses a beam of light onto the
specimen. It can be fixed or movable, to adjust the quality of light, but this entirely
depends on the microscope.
 They are held together by a sturdy metallic curved back used as an arm and a stand at
the bottom, known as the base, of the microscope. The arm and the base hold all the
parts of the microscope.
  The stage where the specimen is placed, allowing movement of the specimen around
for better viewing with the flexible knobs and it is where the light is focused on.
 Two focusing knobs i.e the fine adjustment knob and the coarse adjustment knob, found
on the microscopes’ arm, which can move the stage or the nosepiece to focus on the
image. the sharpen the image clarity.
 It has a light illuminator or a mirror found at the base or on the microbes of the
nosepiece.
 The nosepiece has about three to five objective lenses with different magnifying power.
It can move round to any position depending on the objective lens to focus on the image.
 An aperture diaphragm also is known as the contrast, which controls the diameter of
the beam of light that passes through the condenser, in that, when the condenser is
almost closed, the light comes through to the center of the condenser creating high
contrast. But when the condenser is widely open, the image is very bright with very low
contrast.
Magnification by Bright field Microscope (Compound light
microscope)
During visualization, the objective lens remains parfocal which means, when the objective lens
is changed, the image still remains in focus. The objective lens plays a major role in focusing
the image on the condenser forming an enlarged clear image within the microscope, which is
then further magnified by the eyepiece to a primary image.
What is seen in the microscope as an enlarged clear image of the specimen is known as the
virtual image. To calculate the magnification, multiply the objective and eyepiece objective
magnification together. The magnification is standard, i.e not too high nor too low, and
therefore depending on the magnification power of the lenses, it will range between 40X and
100oX.
Calculation of magnification = Magnification of objective lens/magnification of the eyepiece
lens
The objective lens plays a vital role in not only enlarging the image but also making it clear for
viewing, a feature known as resolution. Resolution according to Prescott, is the ability of a
lens to separate or distinguish between small objects closely linked together.
Whereas the eyepiece magnifies the image at the end of the viewing, its magnification range is
lower than that of the objective lens at 8X-12X (10X standard) and that of the objective lens at
40X-100X, magnification, and resolution of the microscope is highly dependant on the
objective lens.
Applications of the Bright Field Light Microscope (Compound light
microscope)
Vastly used in Microbiology, this microscope is used to view fixed and live specimens, that
have been stained with basic stains. This gives contrast for easy visibility under the microscope.
Therefore it can be used to identify basic bacteria cells and parasitic protozoans such
as Paramecium.

Phase Contrast Microscope

 This is a type of optical microscope whereby small light deviations know as phase
shifts occur during light penetration into the unstained specimen. These phase shifts
are converted into the image to mean, when light passes through the opaque specimen,
the phase shifts brighten the specimen forming an illuminated (bright) image in the
background.
  The phase-contrast microscope produces high contrast images when using a transparent
specimen more so those of microbial cultures, thin tissue fragments, cell tissues, and
subcellular particles.
 The principle behind the working of the phase-contrast microscope is the use of an
optical method to transform a specimen into an amplitude image, that’s viewed by the
eyepiece of the microscope.
 The PCM can be used to view unstained cells also known as the phase objects, which
means that the morphology of the cell is maintained and the cells can be observed in
their natural state, in high contrast and efficient clarity. This is because if the specimens
are stained and fixed, they kill most cells, a characteristic that is uniquely undone by
the brightfield light microscope.
 The shifts that occur during light penetration, become converted to changes in
amplitude which causes the image contrast.
 Coupled with contrast-enhancing elements such as fluorescence, they produce better
visuals of the specimens’ image.
Parts of the Phase Contrast Microscope
The instrumentation of the Phase Contrast Microscope is based on its light pathways from
receiving the source of light to the visualization of the image.
Therefore its sequentially made up of:
 Light source (Mercury arc lamp)
 Collective lens
 Aperture
 Condenser
 Condenser annular
 Specimen
 Objective
 Phase plate
 Deflected light
 Phase ring
The functioning of the Phase Contrast microscope
 The change caused by the deviated scattered (Deflected) light and the undeviated light
that reaches the specimen which is absorbed, create at a certain wavelength, producing
color. The difference created by the scattered light and that of the absorbed light is
known as amplitude variations. These amplitude variations are sensitive to allowing
visualization by photographic equipment like the Phase Contrast Microscope, hence
seen by the human eye.
 The Condenser of the phase-contrast microscope has an opaque disk that is known as
an annular ring, with a transparent ring that produces a cone of light, that passes through
a specimen. Due to light variations some light bend at the specimen, caused by
variations in light density, forming an image at the objective lens. The undeviated light
will strike the phase ring on the phase plate and the deviated light will miss the phase
ring passing through the phase plate directly, this forms an image.
The Phase-Contrast Microscope is designed with objective lenses that have the ability to
perform multiple functions when combined with contrast-enhancing techniques, for example,
fluorescence. The objective lenses are located in the internal phase plate with variation in the
light absorption and phase displacement i.e undiffraction, creating a wide spectrum for
contrasting the specimen and forming a strong contrast in the background.
Applications of Phase-Contrast Microscope
 Determine morphologies of living cells such as plant and animal cells
  Studying microbial motility and structures of locomotion
 To detect certain microbial elements such as the bacterial endospores

Dark-Field Light Microscope

This is a specialized type of bright field light microscope which has several similarities to the
Phase-Contrast Microscope. To make a dark field Microscope, place a darkfield stop
underneath and a condenser lens which produces a hollow cone beam of light that enters the
objective only, from the specimen (Prescott, pg 22).
This technique is used to visualize living unstained cells. This is effected by the way
illumination is done on the specimen in that, when a hollow cone beam of light is transmitted
to the specimen, deviated light (unreflected/unrefracted) rays do not pass through the objectives
but the undeviated (reflected/refracted) light passes through the objectives to the specimen
forming an image.
This makes the surrounding field of the specimen appear black while the specimen will appear
illuminated. This is enabled by the dark background this the name, dark-field Microscopy.
Applications of the Dark Field Microscope
 It is used to visualize the internal organs of larger cells such as the eukaryotic cells
 Identification of bacterial cells with distinctive shapes such as Treponema pallidum, a
causative agent of syphilis.

The Fluorescent Microscope

The above-discussed microscopes will normally produce images after a light has been
transmitted and passed through the specimen.
In the case of the fluorescent Microscope, the specimen emits light. How? By adding a dye
molecule to the specimen. This dye molecule will normally become excited when it absorbs
light energy, hence it releases any trapped energy as light. The light energy that is released by
the excited molecule has a long wavelength compared to its radiating light. The dye molecule
is normally a fluorochrome, that fluoresces when exposed to the light of a certain specific
wavelength. The image formed is a fluorochrome-labeled image from the emitted light
The principle behind this working mechanism is that the fluorescent microscope will expose
the specimen to ultra or violet or blue light, which forms an image of the specimen that is
emanated by the fluorescent light. They have a mercury vapor arc lamp that produces an intense
beam of light that passes through an exciter filter. The exciter filter functions to transmit a
specific wavelength to the fluorochrome stained specimen, producing the fluorochrome-
labeled image, at the objective.
After the objective, there is a barrier filter that functions primarily to remove any ultraviolet
radiation that may be harmful to the viewer’s light, thus reducing the contrast of the image.

Applications of the Fluorescent Microscope

 Used in the visualization of bacterial agents such as Mycobacterium tuberculosis.
 Used to identify specific antibodies produced against bacterial antigens/pathogens in
immunofluorescence techniques by labeling the antibodies with fluorochromes.
 Used in ecological studies to identify and observe microorganisms labeled by the
fluorochromes
 It can also be used to differentiate between dead and live bacteria by the color they emit
when treated with special stains
Besides the above-discussed microscopes, there is one not commonly used microscope known
as the Differential Interference Contrast Microscopy. It is very similar to the phase-contrast
microscope whereby the images are formed from the variations in the light either deviated and
or undeviated. The difference is, here two beams of light are emitted to the specimen and
focused by a prism. One beam passes through the prism to the specimen while another passes
through the glass slide clear area without the specimen. The two beams then combine and
interfere with each other to form an image. It can be used to view cell structures such as
endospores, bacterial cell walls, nuclei and granules for unstained specimens.