EBS 202

Microbiology

Biosystems Engineering

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Introduction to the Science of
Microbiology
-
Methods of Observation and
Microscope

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After learning this lesson you should be able to,
Explain basic principles of microscopic methods
Identify a use for darkfield, phase-contrast,, fluorescence,
confocal microscopy, and compare each with bright field
microscope
Explain how electron microscopy differs from light
microscopy

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Microscopy and the Origins of Microbiology
Microscope is considered as the oldest and most fundamental
instrument used in Microbiology
Microbiology did not exist before the invention of the
microscope

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Microscope
An optical instrument used to magnify minute objects or
microorganisms which cannot be seen by naked eye
The word microscope is derived from the Latin word micro
(small) and the Greek word skopos (to look at)
Basic Purposes of Microscopy in Microbiology

 Initial detection of microbes

 Preliminary or definitive identification of microbes

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Units of Measurement
Microorganisms are measured in smaller units, such as
micrometers and nanometers
A micrometer (μm) equals 0.000001 m (10-6 m)
A nanometer (nm) equals 0.000000001 m (10-9 m)

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Common Units of Measurements
Unit Abbreviation Value

1 centimeter cm 10−2 meter

1 millimeter mm 10−3 meter

1 micrometer µm 10−6 meter

1 nanometer nm 10−9 meter

1 Angstrom Å 10−10 meter

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Development of Microscope
The simple microscope used by van Leeuwenhoek in the
had only one lens
Thus, he became the first person to observe and describe
microorganisms accurately
Robert Hooke, built compound microscopes, which have
multiple lenses
Then, a Dutch spectacle maker, Zaccharias Janssen, is
credited with making the first compound microscope
around 1600

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Cont.
In 1830 a significantly advanced microscope was developed
by Joseph Jackson Lister
Various improvements to Lister’s microscope resulted in the
development of the modern compound microscope

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Magnification
Capacity of a microscope to enlarge an image
𝑇𝑜𝑡𝑎𝑙 𝑀𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑎𝑡𝑖𝑜𝑛
= 𝑂𝑐𝑢𝑙𝑎𝑟 𝑙𝑒𝑛𝑠 𝑀𝑎𝑔𝑖𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛 × 𝑂𝑏𝑗𝑒𝑐𝑡𝑖𝑣𝑒 𝑙𝑒𝑛𝑠 𝑀𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛

In light microscope,

Power of objective Usual power of Magnification
ocular
4× (scanning objective) 10× 40×
10× (low power objective) 10× 100×
40× (high dry objective) 10× 400×
100× (oil immersion objective) 10× 1000× 10
Resolution or Resolving Power
Ability to distinguish two adjacent objects as distinct
and separate
Specifically, it refers to the ability of the lenses to
distinguish two points that are a specified distance apart
The limit of resolution for a light microscope is about 0.2
μm

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Refractive Index
When a ray of light passes from one medium to another,
refraction occurs
The refractive index is a measure of how greatly a particular
medium slows the velocity of light
Thus refractive index measures the light-bending ability of a
medium
When light passes from air into glass (a greater refractive index),
the light ray is slowed and bent toward the normal
As light leaves glass and returns to air (low refractive index),light
accelerates and is bent away from the normal
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Cont.
Light rays move in a straight line through a single medium
After the specimen is stained, the specimen and its medium
have different refractive indexes
This increases the image’s contrast between the specimen
and the medium

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The Use of Immersion Oil
At very high magnifications, resolution may be decreased
when light passes through the small amount of air between
the specimen and the lens
This is due to the large difference between the refractive
indices of air and glass
The air scatters the light rays before they can be focused by
the lens

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Cont.
An optical grade oil known as immersion oil can be placed
between the glass slide and the oil immersion objective lens
Immersion oil has the same refractive index as glass
Immersion oil can increases the light gathering ability of a
lens and increases the amount of light that is collected and
viewed by the lens

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Cont.

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Cont.

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Different Types of Microscopes
1. Light microscopy
 Bright-field (light) microscope
 Dark-field microscopy
 Phase-contrast microscopy
 Fluorescent microscopy
 Confocal microscopy
2. Electron microscopy
 Transmission electron microscopy (TEM)
 Scanning electron microscopy (SEM)
3. Scanning probe microscopy
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Principles of Light Microscopy
A microscope that uses visible light to observe
specimens
In light microscopy, light typically passes through a
specimen and then through a series of magnifying lenses

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Bright-Field Microscope
In bright field microscope,
o Light rays from an illuminator (light source) used to illuminate the
specimen
o Light rays pass through the condenser to the specimen
o The image of the specimen is magnified first by the objective lens
and then by the ocular lens

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Principle Parts of Compound Light Microscope

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https://courses.lumenlearning.com/microbiology/chapter/instruments-of-microscopy/
Cont.
 Light from an illuminator (light source) is focused on the
specimen by the condenser
o Condenser - A lens that focuses the light from the illuminator onto the
specimen
 This light passes through the sample and is collected by the
lenses.
o Objective lenses - The objective lens magnifies the specimen and creates
a real image. Usually microscopes have 3-5 lenses of different
magnifications (ex: 4×, 10×, 40× and 100×)

o Ocular lens (eyepiece) – Ocular lens further magnifies the image

obtained from objective lens and create a virtual image. Typically ocular
lenses magnify an image 10×

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Cont.
Coarse adjustment knob - Moves the stage toward or away
from the objective lens to bring the image into focus at low
power
Fine adjustment knob - used for “fine-tuning” an image.
Fine focus can only be used when the 40X and 100X
objectives are in place

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Improving contrast in Light Microscopy
Simple or differential staining can be used to enhance
contrast.
Specific bacterial structures such as capsules, endospores,
and flagella also can be selectively stained.

Contrast is necessary in light microscopy to

distinguish microorganisms
from their surroundings

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Cont.
 Dyes can be used to stain cells and increase their contrast
 Each class of dye has an affinity for specific cellular materials
 Many dyes used in microbiology are positively charged and they are
called as basic dyes
Ex:
o methylene blue
o crystal violet
o Safranin

 Basic dyes bind strongly to negatively charged cell components, such

as nucleic acids and acidic polysaccharides
 These dyes also stain the surfaces of cells, because cell surfaces tend
to be negatively charged
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The Dark Field Microscopy
A bright-field microscope can be adapted as a dark-field
microscope by adding a special disc to the condenser
A small, opaque disk (about 1 cm in diameter) is placed
between the illuminator and the condenser lens
The disk blocks most of the light from the illuminator as it
passes through the condenser on its way to the objective lens,
producing a hollow cone of light that is focused on the
specimen

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Cont.
The only light that reaches the objective is light that has
been refracted or reflected by structures in the specimen
Because there is no direct background light, the specimen
appears light against a black background—the dark field

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Cont.
The condenser is designed to form a hollow cone of light as
opposed to bright field microscopy that illuminates the
sample with a full cone of light

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Cont.
In dark field microscopy, the objective lens sits in the dark
hollow of this cone and light travels around the objective
lens
The entire field of view appears dark when there is no
sample on the microscope stage
However, when a sample is placed on the stage it appears
bright against a dark background

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Applications of Dark Field Microscopy
Examine live microorganisms that are invisible in the
ordinary light microscope,
Examine unstained microorganisms suspended in liquid

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Phase Contrast Microscopy
Phase-contrast microscopes use refraction and interference
caused by structures in a specimen to create high-contrast,
high-resolution images without staining
This creates an image by altering the wavelengths
of light rays passing through the specimen

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Cont.
To create altered wavelength paths, an annular
ring/annular diaphragm is used in the condenser
The annular ring produces a hollow cone of light that is
focused on the specimen before reaching the objective lens
The objective contains a phase plate containing a phase ring

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 Cont.
The annular ring is an opaque disk with a thin
transparent ring, which produces a hollow
cone of light
Phase plate/ phase ring is located in
the objective lens
Phase plate is a transparent glass disc with
one or few channels (channels are coated with
a material that can absorb light but cannot
retard it)
Rest of the plate is coated with light retarding
materials 33
Cont.
Light traveling directly from the illuminator passes through
the phase ring while light refracted or reflected by the
specimen passes through the plate

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Cont.
This optical technique converts differences in the refractive
index of the sample into shifts in the phase of light and uses
these phase shifts to create contrast
Thus, phase contrast permits visualization of details such as
intracellular organelles

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What Is Light Phase?
If two waves with the same wavelength
start from the same point and take
different paths before reaching the
same point, there will be then a difference in phase between
the two waves
This means that phase differences in light from the same
sample can be introduced by forcing some of the light to
travel further
This is the concept behind phase-contrast

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Cont.
Transparent unstained samples (such as cells) do not absorb
light and are called phase objects
When light passes through a sample area with no phase
object, there is no significant change in the RI so no
significant diffraction occurs
This light that is not diffracted is often referred to
as direct light as it continues unmodified through the
sample

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Cont.
On the other hand, when light passes through an area of the
sample with a phase object (such as cellular structures)
small changes in the RI will diffract and scatter some light
and cause changes to the light path
This depends on the thickness and RI of each structure
The thicker the structure, the greater the diffraction of the
light

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Cont.

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Working Principle
When light rays passes through a area of high refractive
index, its deviates from its normal path and experiences a
phase change
When light rays passes through an area with a less refractive
index, rays remain undeviated. So no phase change
With the help of annular ring and phase ring, the difference
between these phase changes of retarded and un-retarded
light rays can be converted into a brightness change
Thus, a contrast difference can be created in the final image
of the specimen

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Cont.

The same field of cells of the yeast

Saccharomyces cerevisiae visualized by (a) bright-field
microscopy, (b) phase-contrast microscopy
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Applications of Phase Contrast Microscopy

1. To study unstained living cells

2. Detailed examination of internal structures in living
microorganisms.
3. To study flagellar movements and motility of bacteria and
protozoans
4. To study intestinal and other live protozoa such as
amoebae and Trichomonas
5. To examine fungi grown in culture

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Cont.

An illustration of light pathways of bright field, dark field and phase

contrast microscopy 43
 Cont.

bright-field microscopy phase-contrast microscopy dark-field microscopy

Micrographic images Saccharomyces cerevisiae captured from different types

of light microscopes

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Fluorescence Microscopy
 The fluorescence microscope visualizes specimens that fluoresce
 Fluorescence is the ability of substances to absorb short
wavelengths of light (ultraviolet) and give off light at a longer
wavelength (visible)
 This is a specially modified compound microscope equipped
with an ultraviolet (UV) radiation source and a filter to protects
the viewer’s eye from dangerous rays
 Cells fluoresce either because they,
o contain naturally fluorescent substances such as chlorophyll or
o have been stained with a fluorescent dye
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Cont.
Fluorescent dyes are also called as fluorochromes
If cells or bacteria are stained with a fluorescent dye are
examined under the microscope with ultraviolet light
instead of ordinary visible light, they become luminous and
are seen as bright objects against a dark background

Escherichia coli stained with

fluorescent antibodies (600x) 46
Cont.
Fluorochromes have special attractions for different
microorganisms
For example, the fluorochrome auramine O, which glows
yellow when exposed to ultraviolet light, is strongly
absorbed by Mycobacterium tuberculosis
When the dye is applied to a sample of material suspected
of containing the bacterium, the bacterium can be detected
by the appearance of bright yellow organisms against a dark
background

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Cont.
DAPI (4’,6-diamidino-2-phenylindole) is a widely used
fluorescent dye
This dye stains cells bright blue by making complexes with
the cell’s DNA
DAPI can be used to visualize cells in their natural habitats,
such as soil, water, food, or a clinical specimen

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Applications of Fluorescence Microscopy
Fluorescence microscopy using DAPI is widely used in
clinical diagnostic microbiology and also in microbial
ecology for enumerating bacteria in a natural environment
or in a cell suspension

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Cont.
The principal use of fluorescence microscopy is a diagnostic
technique called the fluorescent-antibody (FA) technique,
or immunofluorescence
Antibodies are natural defense molecules that are produced
by humans and many animals in reaction to a foreign
substance, or antigen

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Cont.
Fluorescent antibodies for a particular antigen are obtained
as follows:
o An animal is injected with a specific antigen, such as a bacterium
o Then the animal begins to produce antibodies against that antigen
o After a sufficient time, the antibodies are removed from the serum
of the animal
Next, a fluorochrome is chemically combined with the
antibodies

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Cont.
These fluorescent antibodies are then added to a microscope slide
containing an unknown bacterium
If this unknown bacterium is the same bacterium that was injected
into the animal, the fluorescent antibodies bind to antigens on the
surface of the bacterium, causing it to fluoresce

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Confocal Microscopy
Confocal microscopy is a computer-controlled microscope
In addition to the fluorescence microscopy, this has a laser source
The laser generates a high-contrast, three-dimensional image and
allows the viewer to access several planes of focus in the specimen

Confocal
Microcopy 53
Cont.
Fluorescently stained specimens are usually examined
Often, the specimens are first stained or tagged with a
fluorescent dye
A computer is used to assembles the data and constructs a
three-dimensional image, which is displayed on a screen

Confocal image of a filamentous

cyanobacterium 54
Applications of Confocal Microscopy
To obtain three-dimensional images of entire cells and
cellular components
To evaluate cellular physiology—by monitoring the
distributions and concentrations of substances such as ATP
To observe thick specimens

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 Electron Microscopy
Electron microscope uses electromagnetic lenses, electrons,
and a fluorescent screen to produce the magnified image
Electrons are used instead of visible light (photons) to image
cells and cell structures
The system of electron microscope operates in a vacuum
The image can be captured on photographic film to create an
electron photomicrograph

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Cont.
 Electron microscopy has a superior resolution
 The better resolution of electron microscopes is due to the
shorter wavelengths of electrons
 The wavelengths of electrons are about 100,000 times smaller
than the wavelengths of visible light
 The electron beam is focused by electromagnetic lenses (similar
to glass lenses in the light microscope)
 The object which is held in the path of the beam scatters the
electrons and produces an image which is focused on a
fluorescent viewing screen

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Cont.
Images produced by electron microscopes are always black
and white, but they may be colored artificially to highlight
certain details
There are two types
o transmission electron microscope
o scanning electron microscope

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The Transmission Electron Microscope (TEM)
This microscope produces its image by transmitting electrons
through the specimen
The transmission electron microscope has a practical resolution
roughly 1,000 times better than the light microscope
In many electron microscopes, points closer
than 0.5 nm can be distinguished, and the
useful magnification is well over 100,000

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Cont.
In the TEM, a finely focused beam of electrons from an
electron gun passes through a specially prepared, ultrathin
section of the specimen
The beam is focused on a small area of the specimen by an
electromagnetic condenser lens
Instead of being placed on a glass slide, as in light
microscopes, the specimen is usually placed on a copper
mesh grid

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Cont.
The beam of electrons passes through the specimen and
then through an electromagnetic objective lens, which
magnifies the image
Finally, the electrons are focused by an electromagnetic
projector lens (rather than by an ocular lens as in a light
microscope) onto a viewing screen and saved as a digital
image
The final image, called a transmission electron micrograph
This appears as many light and dark areas, depending on the
number of electrons absorbed by different areas of the
specimen
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Sample preparation for TEM
Electrons are very poor at penetrating, even a single cell is
too thick to penetrate with an electron beam
Consequently, to view the internal structure of a cell, thin
sections of the cell are needed, and the sections must be
stabilized and stained with various chemicals to make them
visible
Contrast can be greatly enhanced by using a “dye” that
absorbs electrons and produces a darker image in the
stained region
Salts of various heavy metals, such as lead, osmium,
tungsten, and uranium, are commonly used as stains
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 Cont.

Electron micrograph of a thin section of a dividing bacterial cell,

taken by transmission electron microscopy

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Cont.

TEM section through a cell of Chlamydomonas 64

Cont.
Transmission electron microscopy has high resolution and
is extremely valuable for examining different layers of
specimens
However, it does have certain disadvantages
Electrons have limited penetrating power, only a very thin
section of a specimen (about 100 nm) can be studied
effectively.
Thus, the specimen has no three-dimensional aspect
In addition, specimens must be fixed, dehydrated, and
viewed under a high vacuum to prevent electron scattering
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TEM

https://www.newcastle.edu.au/research/facilities/central-
analytical-facilities/emx/tem 66
The Scanning Electron Microscope
The SEM produce an image from electrons emitted by an
object’s surface rather than from transmitted electrons
In scanning electron microscopy, the specimen is coated
with a thin film of a heavy metal, typically gold
 An electron beam then scans back and forth across the
specimen
Electrons scattered from the metal coating are collected and
projected on a monitor to produce an image

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Cont.
In the SEM, even fairly large specimens can be observed
SEM can resolve objects as close together as 10 nm, and
objects are generally magnified 1000 to 500,000 x
However, only the surface of an
object is typically visualized

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Sample Preparation for SEM
Specimen preparation is easy, and in some cases air-dried
material can be examined directly
Yet, microorganisms have to be fixed, dehydrated, and dried
first, to preserve surface structure and prevent collapse of
the cells when they are exposed to the SEM’s high vacuum
Before viewing, dried samples are mounted and coated with
a thin layer of metal to prevent the buildup of an electrical
charge on the surface and to give a better image

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Cont.

Scanning electron micrograph (SEM) of bacterial

cells

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Cont.

SEM (left) and TEM (right) images of bacteria. Whereas SEM shows numerous bacteria
on a surface (green), the TEM image shows the interior structure of a single bacterium.
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Scanning Probe Microscopy
This microscope measure surface features by moving a
sharp probe over the object’s surface
It can achieve magnifications of 100 million and allow
scientists to view atoms on the surface of a solid
A tip moves up and down while following the surface
contours of a specimen, its motion is recorded and analyzed
by a computer to create an accurate three dimensional
image of the surface atoms
Resolving power is much greater than the electron
microscope

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Applications of Scanning Probe Microscopy
Use to observe incredibly detailed views of molecules such
as DNA

Image of DNA from Scanning

Probe Microscopy
(DNA double helix with
approximately three turns
shown) x2,000,000). 73
References

Thomas D. Brock, Michael T. Madigan: Biology of

microorganisms, 6th Edition. Englewood Cliffs, N.J.,
Prentice-Hall, 1991.
https://www.photometrics.com/learn/microscopy-
basics/phase-contrast-microscopy

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Thank You

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