BY YASA MWELWA

UNIT 1: INTRODUCTION TO
BIOLOGY
• The word "biology" is derived from the Greek words
"bios" (meaning life) and "logos" (meaning "study").
Biology is the study of life.
• Biology is the scientific study of life. It is a natural
science with a broad scope but has several unifying
themes that tie it together as a single, coherent field.
• The study of living organisms, divided into many
specialized fields that cover their morphology,
physiology, anatomy, behavior, origin, and distribution
• What makes something ―alive? Anyone could deduce
that a galloping horse is alive and a car is not, but why?
 BY YASA MWELWA

CHARACTERISTICS OF LIVING
ORGANISMS
• Cellular organization - All organisms consist of one or more cells.
• Ordered complexity - All living things are both complex and highly ordered. Your body
is composed of many different kinds of cells, each containing many complex molecular
structures.
• Sensitivity - All organisms respond to stimuli. Plants grow toward a source of light, and
the pupils of your eyes dilate when you walk into a dark room
• Growth, development, and reproduction - All organisms are capable of growing and
reproducing, and they all possess hereditary molecules that are passed to their
offspring
• Energy utilization - All organisms take in energy and use it to perform many kinds of
work.
• Homeostasis - All organisms maintain relatively constant internal conditions that are
different from their environment, a process called homeostasis. For example, your body
temperature remains stable despite changes in outside temperatures.
• Evolutionary adaptation - All organisms interact with other organisms and the non-
living environment in ways that influence their survival, and as a consequence,
organisms evolve adaptations to their environments
 BY YASA MWELWA

BIOLOGICAL SCIENCES EMBRACE

THE FIELDS OF:
• Zoology – science of animals
• A branch of zoology specializing in the study of insects
is termed Entomology.
• Another branch of zoology dealing with parasitic
animals is called Parasitology
• The science of plants is known as Botany or Phytology
• The study of fungi is termed as Mycology
• The study of Bacteria is covered in the discipline of
biology called Bacteriology
• The special study of viruses is termed Virology
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• The fields of bacteriology, mycology and virology

constitute a major discipline of biology called Microbiology
- Microbiology is the study of microscopic organisms
(bacteria, fungi, viruses)
• The branch of biology dealing with heredity and biological
variation is called Genetics.
• Ecology – the study that involves the interactions between
organisms (plants and animals) and their environment.
• Biology can be studied from several angles that include:
a) Morphology – a field which deals with the form and
structure of organisms
b) Anatomy – a field that involves the study of the internal
structures of organs and associated tissue types
 BY YASA MWELWA

MICROSCOPY
• The cells are so minute that they can only be
studied under the microscope.
• A microscope is a device that allows people to
view specimens in detail too small for the naked
eye to see. They do this by magnification and
resolution.
• Magnification is how many times the object is
enlarged within the viewing lens. Resolution is
how detailed the object appears when viewed.
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• The early pioneers of microscopy opened a

window into the invisible world of
microorganisms. But microscopy continued to
advance in the centuries that followed.
• The field of biology emerged from the
development of the microscope, and the new
found ability to see microorganisms.
 BY YASA MWELWA

MICROSCOPE USES/APPLICATIONS
 BY YASA MWELWA

HISTORY OF MICROSCOPY
• Although scientifically, the first simple microscope was
discovered by two Dutch scientists, Zacharias Janssen
and his father, Hans Janssen.
• Dutch father-son team named Hans and Zacharias
Janssen invented the first so-called compound
microscope in the late 16th century when they
discovered that, if they put a lens at the top and
bottom of a tube and looked through it, objects on the
other end became magnified.
• Despite not being included as a scientific discovery, this
act paved the way for scientific evolution.
BY YASA MWELWA
 BY YASA MWELWA

HISTORY OF THE DEVELOPMENT

OF MICROSCOPES

1. ROBERT HOOKE
• English natural philosopher Robert Hooke first
described cells in 1665. When he used a
microscope he had built to examine a thin slice of
a non-living tissue found in the bark of certain
trees.
• Hooke observed a honeycomb of tiny, empty
(because the cells were dead) compartments. He
called the compartments cellulae (Latin, “small
rooms”), and the term has come down to us as
“cells”
BY YASA MWELWA
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2. ANTONIE VAN LEEUWENHOEK

• The first living cells were observed a few years later by the
Dutch naturalist Antonie van Leeuwenhoek, who called the
tiny organisms that he observed ―animalcules” meaning little
animals.
• Antonie van Leeuwenhoek, sometimes hailed as ―the Father
of Microbiology, is typically credited as the first person to
have created microscopes powerful enough to view microbes.
• Antonie van Leeuwenhoek (1632–1723) is credited as being
the first person to observe microbes, including bacteria, which
he called ―animalcules and ―wee little beasties. Even though
van Leeuwenhoek’s microscopes were simple microscopes (as
seen in this replica), they were more powerful and provided
better resolution than the compound microscopes of his day.
BY YASA MWELWA
 BY YASA MWELWA

Antonie van Leeuwenhoek’s

microscope
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BEGINNING OF THE CELL THEORY

3. MATTHIAS SCHLEIDEN
• For another century and a half, however, biologists
failed to recognize the importance of cells. In 1838, a
German botanist named Matthias Schleiden concluded
that all plants were made of cells.
• Schleiden is a co-founder of the cell theory made a
careful study of plant tissues and developed the first
statement of the cell theory.
• He stated that: ―all plants are aggregates of fully
individualized, independent, separate beings, namely
the cells themselves.
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4. THEODORE SCHWANN
• In 1839, a German zoologist named Theodore
Schwann concluded that all animals were
made of cells. Schwann also co-founded the
cell theory.
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5. RUDOLPH VIRCHOW
• In 1855, a German medical doctor named
Rudolph Virchow observed, under the
microscope, cells dividing.
• He reasoned that all cells come from other
pre-existing cells by cell division.
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THE CELL THEORY

• Explains relationship between cells and living things –
foundation of modern biology
• Cell theory is a collection of ideas and conclusions from
many different scientists over time that describes cells
and how cells operate.
• The cell theory, in its modern form, includes the
following three principles:
• 1. All organisms are composed of one or more cells, and
the life processes of metabolism and heredity occur
within these cells.
• 2. Cells are the smallest basic units of living things, which
make the structural organization of all organisms.
• 3. Cells arise only by cell division of a previously existing
cells.
BY YASA MWELWA
 BY YASA MWELWA

MAGNIFICATION, RESOLUTION,
AND CONTRAST
• Microscopes magnify images and use the
properties of light to create useful images of
small objects.
• Magnification is defined as the ability of a
lens to enlarge the image of an object when
compared to the real object. For example, a
magnification of 10⨯ means that the image
appears 10 times the size of the object as
viewed with the naked eye.
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• Greater magnification typically improves our ability to see

details of small objects, but magnification alone is not
sufficient to make the most useful images.
• It is often useful to enhance the resolution of objects: the
ability to tell that two separate points or objects are
separate. A low-resolution image appears fuzzy, whereas a
high-resolution image appears sharp. Two factors affect
resolution.
• The first is wavelength. Shorter wavelengths are able to
resolve smaller objects; thus, an electron microscope has a
much higher resolution than a light microscope, since it
uses an electron beam with a very short wavelength, as
opposed to the long-wavelength visible light used by a light
microscope.
• The second factor that affects resolution is numerical
aperture, which is a measure of a lens’s ability to gather
light. The higher the numerical aperture, the better the
resolution.
 BY YASA MWELWA

• Even when a microscope has high resolution, it

can be difficult to distinguish small structures in
many specimens because microorganisms are
relatively transparent. It is often necessary to
increase contrast to detect different structures in
a specimen.
• Various types of microscopes use different
features of light or electrons to increase
contrast—visible differences between the parts
of a specimen.
• Additionally, dyes that bind to some structures
but not others can be used to improve the
contrast between images of relatively transparent
objects.
 BY YASA MWELWA

• There are several types of microscopes – they

include: light, electron, and scanned-probe
microscopes.
• Light microscopes view cells at a low
magnification
• Electron and scanned-probe are able to view
cells and cell structures at a very high
magnification.
 BY YASA MWELWA

1. Compound light microscopy uses visible light to

illuminate specimens. Types used in microbiology
include: Brightfield, phase-contrast, differential
interference contrast (DIC), dark-field, fluorescence,
and confocal.
2. Electron microscopy uses electrons instead of
visible light for illumination. Types are: Transmission
electron microscopy (TEM) and scanning electron
microscopy (SEM).
3. Scanned-probe microscopy uses probes to
examine surfaces of specimens. Example: Atomic
force microscopy (AFM) and Scanning Tunneling
Microscopy (STM):
 BY YASA MWELWA

Some key terms:

• Magnification: Ratio of a specimen’s image

size to its real size.
• Total magnification: The product of
magnification of ocular and objective lenses.
• Resolution: The minimum distance that
distinguishes (or the ability to distinguish) the
separation between two closely located
objects. Resolution is the limiting factor in the
ability to see small objects.
 BY YASA MWELWA

DETERMINING RESOLUTION:
• Resolution is function of the wavelength of light
and the numerical aperture of the objective lens
– resolution and wavelength are inversely related.
• Therefore, the shorter the wavelength the greater
the resolving power.
• (Resolution) Resolving power, d = 0.5ƛ/NA
• Numerical aperture (NA) of the lens its ability to
bend or refract light passing through it.
• ƛ = wavelength
 BY YASA MWELWA

TYPES OF MICROSCOPES
 BY YASA MWELWA

• 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
 BY YASA MWELWA

1.BRIGHTFIELD MICROSCOPE
 BY YASA MWELWA

BRIGHTFIELD 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.
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BRIGHTFIELD MICROSCOPE:

• Some bright-field microscopes are monocular (having a

single eyepiece), though most newer bright-field
microscopes are binocular (having two eyepieces); in
either case, each eyepiece contains a lens called an ocular
lens.

• The ocular lenses typically magnify images 10 times

(10⨯). At the other end of the body tube are a set of
objective lenses on a rotating nosepiece. The
magnification of these objective lenses typically ranges
from 4⨯ to 100⨯, with the magnification for each lens
designated on the metal casing of the lens.
 BY YASA MWELWA

• The ocular and objective lenses work together

to create a magnified image. The total
magnification is the product of the ocular
magnification times the objective
magnification:
• For example, if a 40⨯ objective lens is
selected and the ocular lens is 10⨯, the total
magnification would be:
• (40×)(10×) = 400×
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• The item being viewed is called a specimen. The specimen

is placed on a glass slide, which is then clipped into place
on the stage (a platform) of the microscope.
• Once the specimen is centered over the light, the stage
position can be raised or lowered to focus the image.
• The coarse focusing knob is used for large-scale
movements with 4⨯ and 10⨯ objective lenses;
• the fine focusing knob is used for small-scale movements,
especially with 40⨯ or 100⨯ objective lenses.
• Coarse adjustment: Brings the specimen into general
focus.
• Fine adjustment: Fine tunes the focus and increases the
detail of the specimen.
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• In a bright-field microscope, this light is provided by an

illuminator, which is typically a high-intensity bulb
below the stage.
• Light from the illuminator passes up through
condenser lens (located below the stage), which
focuses all of the light rays on the specimen to
maximize illumination.
• If less-than-maximal light levels are needed, the amount
of light striking the specimen can be easily
adjusted by opening or closing a diaphragm between
the condenser and the specimen.
• In some cases, brightness can also be adjusted using
the rheostat, a dimmer switch that controls the intensity
of the illuminator.
 BY YASA MWELWA

• A bright-field microscope creates an image by

directing light from the illuminator at the
specimen; this light is differentially
transmitted, absorbed, reflected, or refracted
by different structures of the specimen.
• In general, structures in the specimen will
appear darker against a bright background,
creating maximally sharp images at
magnifications up to about 1000⨯.
• This allows us to see objects as small as
bacteria, which are visible at about 400⨯ or so,
but not smaller objects such as viruses.
 BY YASA MWELWA

• At very high magnifications, resolution may be

compromised 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.
• To solve this problem, a drop of oil can be used
to fill the space between the specimen and an oil
immersion lens, a special lens designed to be
used with immersion oils.
 BY YASA MWELWA

• oil has a refractive index very similar to that of

glass, it increases the maximum angle at which
light leaving the specimen can strike the lens.
This increases the light collected and, thus,
the resolution of the image.
 BY YASA MWELWA

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.
BY YASA MWELWA
 BY YASA MWELWA

2.PHASE-CONTRASTMICROSCOPE
 BY YASA MWELWA

PHASE-CONTRASTMICROSCOPE
• Phase-contrast microscopes use refraction and
interference caused by structures in a specimen to
create high-contrast, high-resolution images without
staining.
• type of microscope that creates an image by altering
the wavelengths of light rays passing through the
specimen. To create altered wavelength paths, an
annular stop is used in the condenser.
• The annular stop 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.
 BY YASA MWELWA

• As a result, light traveling directly from the

illuminator passes through the phase ring
while light refracted or reflected by the
specimen passes through the plate.
• This causes waves traveling through the ring to
be about one-half of a wavelength out of phase
with those passing through the plate.
 BY YASA MWELWA

• Structures that refract light then appear dark

against a bright background of only un-
refracted light.
• Because it increases contrast without requiring
stains (fixation and staining often distort the
shape of cells), phase-contrast microscopy is
often used to observe live specimens.
• Certain structures, such as organelles in
eukaryotic cells and endospores in prokaryotic
cells, are especially well visualized with phase-
contrast microscopy
 BY YASA MWELWA

This figure compares a bright-field image (left) with a phase-

contrast image (right) of the same unstained simple squamous
epithelial cells
 BY YASA MWELWA

• 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 entry, 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.
 BY YASA MWELWA

HOW PHASE CONTRAST PRODUCES AN

IMAGE
• The change is caused by the deviated scattered
(Deflected) light and the undeviated light that
reaches the specimen which is absorbed, created
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.
 BY YASA MWELWA

• 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 bends 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.
 BY YASA MWELWA

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
 BY YASA MWELWA

3. DARKFIELD MICROSCOPE
 BY YASA MWELWA

DARKFIELD MICROSCOPE
• This is a specialized type of bright field light
microscope that 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
• This technique is used to visualize living
unstained cells.
 BY YASA MWELWA

• This is affected 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.
 BY YASA MWELWA

DARKFIELD MICROSCOPE
• A dark-field microscope is a bright-field
microscope that has a small but significant
modification to the condenser.
• A small, opaque disk (about 1 cm in diameter) is
placed between the illuminator and the condenser
lens.
• This opaque light stop, as the disk is called, 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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• The only light that reaches the objective is light

that has been refracted or reflected by structures
in the specimen.
• The resulting image typically shows bright
objects on a dark background.
• Dark-field microscopy can often create high-
contrast, high-resolution images of specimens
without the use of stains, which is particularly
useful for viewing live specimens that might be
killed or otherwise compromised by the stains.
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• For example, thin spirochetes like Treponema

pallidum, the causative agent of syphilis, can
be best viewed using a dark-field microscope
 BY YASA MWELWA

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.
 BY YASA MWELWA

a) Brightfield

b) Phase-contrast

c) Dark-field
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ELECTRON MICROSCOPE

Transmission Electron Microscope Scanning Electron Microscope

 BY YASA MWELWA

ELECTRON MICROSCOPE
• EMs can resolve subcellular structures as well
as some molecular structures (e.g., single
strands of DNA); however, electron
microscopy cannot be used on living material
because of the methods needed to prepare
the specimens.
• There are two basic types of EM: the
transmission electron microscope (TEM) and
the scanning electron microscope (SEM)
 BY YASA MWELWA

• TEM However uses an electron beam from

above the specimen that is focused using a
magnetic lens (rather than a glass lens) and
projected through the specimen onto a
detector.
• Electrons pass through the specimen, and
then the detector captures the image
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4. Transmission electron microscope

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HOW IT WORKS
• You have to prepare a thin slice of the specimen
quite carefully (it's a fairly laborious process)
• When you've done that, you fire an electron
beam down through the specimen from a giant
electron gun at the top.
• The gun uses electromagnetic coils and high
voltages (typically from 50,000 to several million
volts) to accelerate the electrons to very high
speeds.
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• Having reached top speed, the electrons zoom

through the specimen and out the other side,
where more coils focus them to form an image
on screen (for immediate viewing) or on a
photographic plate (for making a permanent
record of the image).
• TEMs are the most powerful electron
microscopes: we can use them to see
INTERNAL STRUCTURES OF ORGANISMS, so
they effectively magnify by a million times or
more.
BY YASA MWELWA
 BY YASA MWELWA

STEP BY STEP PROCESS OF HOW IT

WORKS
• A transmission electron microscope fires a beam of
electrons through a specimen to produce a magnified
image of an object.
• A high-voltage electricity supply powers the cathode.
• The cathode is a heated filament, a bit like the electron
gun in an old-fashioned cathode-ray tube (CRT) TV. It
generates a beam of electrons that works in an analogous
way to the beam of light in an optical microscope.
• An electromagnetic coil (the first lens) concentrates the
electrons into a more powerful beam.
• Another electromagnetic coil (the second lens) focuses the
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beam onto a certain part of the specimen.

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• The specimen sits on a copper grid in the middle of

the main microscope tube. The beam passes
through the specimen and "picks up" an image of it.
• The projector lens (the third lens) magnifies the
image.
• The image becomes visible when the electron beam
hits a fluorescent screen at the base of the machine.
This is analogous to the phosphor screen at the
front of an old-fashioned TV .
• The image can be viewed directly (through a
viewing portal), through binoculars at the side, or
on a TV monitor attached to an image intensifier
(which makes weak images easier to see).
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5. Scanning electron microscope (SEM)

 BY YASA MWELWA

Scanning electron microscopes (SEM)

• (SEM), are designed to make images of the
surfaces of tiny objects.
• Just as in a TEM, the top of a SEM has a
powerful electron gun that shoots an electron
beam down at the specimen.
• A series of electromagnetic coils pull the beam
back and forth, scanning it slowly and
systematically across the specimen's surface.
 BY YASA MWELWA

• The electrons that are reflected off the

specimen (known as secondary electrons) are
directed at a screen, where they create a TV-
like picture.
• SEMs are generally about 10 times less
powerful than TEM
• On the plus side, they produce very sharp, 3D
images (compared to the flat images produced
by TEMs) and their specimens need less
preparation.
BY YASA MWELWA
 BY YASA MWELWA

How a scanning electron microscope

(SEM) works
• A scanning electron microscope scans a beam
of electrons over a specimen to produce a
magnified image of an object.
• Electrons are fired into the machine.
• A positively charged electrode (anode) attracts
the electrons and accelerates them into an
energetic beam.
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• An electromagnetic coil brings the electron

beam to a very precise focus, much like a lens.
• Another coil, lower down, steers the electron
beam from side to side.
• The beam systematically scans across the object
being viewed. Electrons from the beam hit the
surface of the object and bounce off it.
• A detector registers these scattered electrons
and turns them into a picture.
• A hugely magnified image of the object is
displayed on a TV screen.
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BY YASA MWELWA
BY YASA MWELWA
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6. The Fluorescent Microscope

 BY YASA MWELWA

• In the case of the fluorescent Microscope, the

specimen emits light 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
 BY YASA MWELWA

• 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.
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• 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.
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BY YASA MWELWA
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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 fluorochrome.
• Used in ecological studies to identify and observe
microorganisms labeled by the fluorochrome
• It can also be used to differentiate between dead
and live bacteria by the color they emit when
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treated with special stains

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7. Confocal Scanning Laser

Microscope (CSLM)
 BY YASA MWELWA

Confocal Scanning Laser

Microscope (CSLM)
• 2D and 3D. Suitable for thick samples.
• Uses laser (K, Ar, IR) as light source (single- or
multiphoton) to excite fluorescent
• stained specimens, and a pinhole aperture that
eliminates unfocused light.
• One or more lasers illuminate one plane of focus
at a time and an image is recorded (e.g.
• every 0.6μm). A complete image is reconstructed
by the combining of all images recorded.
 BY YASA MWELWA

8. Differential interference contrast

(DIC) microscope
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Differential interference contrast

(DIC) microscope
• (also known as Nomarski optics) are similar to phase-contrast
microscopes in that they use interference patterns to enhance
contrast between different features of a specimen.
• In a DIC microscope, two beams of light are created in which
the direction of wave movement (polarization) differs.
• Once the beams pass through either the specimen or
specimen-free space, they are recombined and effects of the
specimens cause differences in the interference patterns
generated by the combining of the beams.
• This results in high-contrast images of living organisms with a
three-dimensional appearance. These microscopes are
especially useful in distinguishing structures within live,
unstained specimens.
BY YASA MWELWA
BY YASA MWELWA
 BY YASA MWELWA

9. ATOMIC FORCE MICROSCOPE

 BY YASA MWELWA

ATOMIC FORCE MICROSCOPE

• 3D; resolution and working range in the order
of nm to pm.
• Uses a stylus (metal or diamond tip) with
silicon arm Which moves back and forth over
the specimen with movements detected by a
laser.
• Changes or deviations from the starting point
are reconstructed as an image.
BY YASA MWELWA
 BY YASA MWELWA

10. Scanning Tunneling Microscope

 BY YASA MWELWA

Scanning Tunneling Microscope

• Uses an ultrafine tungsten tip.
• Primarily for viewing structures at molecular
and atomic level.
• Tip hovers over a specimen without contact
and allowing currents through its electron
cloud to interact with surface charges of the
specimen.
• The signal generated along the pathway of the
tip is translated into an image
 BY YASA MWELWA

Scanning Tunneling Microscope

 BY YASA MWELWA

STAINING
• In their natural state, most of the cells and
microorganisms that we observe under the
microscope lack color and contrast.
• This makes it difficult, if not impossible, to
detect important cellular structures and their
distinguishing characteristics without
artificially treating specimen.
 BY YASA MWELWA

PREPARING SPECIMENS FOR

LIGHT MICROSCOPY
• In clinical settings, light microscopes are the
most commonly used microscopes. There are
two basic types of preparation used to view
specimens with a light microscope:
1. Wet Mounts
2. fixed specimens (Heat Fixation)
 BY YASA MWELWA

• The simplest type of preparation is the wet

mount, in which the specimen is placed on the
slide in a drop of liquid.
• Some specimens, such as a drop of urine, are
already in a liquid form and can be deposited on
the slide using a dropper. Solid specimens, such
as a skin scraping, can be placed on the slide
before adding a drop of liquid to prepare the wet
mount.
• Sometimes the liquid used is simply water, but
often stains are added to enhance contrast. Once
the liquid has been added to the slide, a coverslip
is placed on top and the specimen is ready for
examination under the microscope.
 BY YASA MWELWA

• The second method of preparing specimens

for light microscopy is fixation. The ―fixing‖
of a sample refers to the process of attaching
cells to a slide.
• Fixation is often achieved either by heating
(heat fixing) or chemically treating the
specimen.
• In addition to attaching the specimen to the
slide, fixation also kills microorganisms in the
specimen, stopping their movement and
metabolism while preserving the integrity of
their cellular components for observation.
 BY YASA MWELWA

• To heat-fix a sample, a thin layer of the

specimen is spread on the slide (called a
smear), and the slide is then briefly heated
over a heat source.
• Chemical fixatives are often preferable to heat
for tissue specimens.
• Chemical agents such as acetic acid, ethanol,
methanol, formaldehyde (formalin), and
glutaraldehyde can denature proteins, stop
biochemical reactions, and stabilize cell
structures in tissue samples.
 BY YASA MWELWA

TYPES OF STAINS (DYES)

• In addition to fixation, staining is almost always
applied to color certain features of a specimen
before examining it under a light microscope.
• Stains, or dyes, contain salts made up of a positive
ion or a negative ion.
• Depending on the type of dye, the positive or the
negative ion may be the chromophore (the colored
ion); the other, uncolored ion is called the counter
ion.
• If the chromophore is the positively charged ion,
the stain is classified as a Basic dye; if the negative
ion is the chromophore, the stain is considered an
Acidic dye.
 BY YASA MWELWA

• Dyes are selected for staining based on the

chemical properties of the dye and the specimen
being observed, which determine how the dye
will interact with the specimen.
• In most cases, it is preferable to use a positive
stain, a dye that will be absorbed by the cells or
organisms being observed, adding color to
objects of interest to make them stand out
against the background.
• However, there are scenarios in which it is
advantageous to use a negative stain, which is
absorbed by the background but not by the cells
or organisms in the specimen. Negative staining
produces an outline or silhouette of the
organisms against a colorful background.
 BY YASA MWELWA

• Because cells typically have negatively charged

cell walls, the positive chromophores in basic
dyes tend to stick to the cell walls, making
them positive stains.
• Thus, commonly used basic dyes such as basic
fuchsin, crystal violet, malachite green,
methylene blue, and safranin typically serve as
positive stains.
• On the other hand, the negatively charged
chromophores in acidic dyes are repelled by
negatively charged cell walls, making them
negative stains. Commonly used acidic dyes
include acid fuchsin, eosin, and rose bengal.
 BY YASA MWELWA

• Some staining techniques involve the application of only

one dye to the sample; others require more than one dye.
• In Simple staining, a single dye is used to emphasize
particular structures in the specimen. A simple stain will
generally make all of the organisms in a sample appear to
be the same color, even if the sample contains more than
one type of organism.
• In contrast, Differential staining distinguishes organisms
based on their interactions with multiple stains. In other
words, two organisms in a differentially stained sample
may appear to be different colors.
• Differential staining techniques commonly used in clinical
settings include Gram staining, acid-fast staining,
endospore staining, flagella staining, and capsule staining.
 BY YASA MWELWA

GRAM STAIN PROCEDURE

• The GRAM STAIN PROCEDURE is a differential
staining procedure that involves multiple steps.
• It was developed by Danish microbiologist Hans
Christian Gram in 1884 as an effective method to
distinguish between bacteria with different types
of cell walls, and even today it remains one of the
most frequently used staining techniques.
• Gram-staining is a differential staining technique
that uses a primary stain and a secondary
counterstain to distinguish between gram-positive
and gram-negative bacteria.
• The steps of the Gram stain procedure are listed
below :
 BY YASA MWELWA

1. First, crystal violet, a Primary Stain, is applied

to a heat-fixed smear, giving all of the cells a
purple color.
• 2. Next, Gram’s iodine, a Mordant, is added. A
mordant is a substance used to set or stabilize
stains or dyes; in this case, Gram’s iodine acts
like a trapping agent that complexes with the
crystal violet, making the crystal violet–iodine
complex clump and stay contained in thick
layers of peptidoglycan in the cell walls.
• it forms Crystal Violet Iodine complex which
increase the intensity of cell to hold the stain.
 BY YASA MWELWA

3. Next, a Decolorizing Agent is added, usually ethanol or

an acetone/ethanol solution. Cells that have thick
peptidoglycan layers in their cell walls are much less
affected by the decolorizing agent; they generally retain
the crystal violet dye and remain purple. However, the
decolorizing agent more easily washes the dye out of cells
with thinner peptidoglycan layers, making them again
colorless.

4. Finally, a secondary Counter-Stain, or Basic fuchsin is

added, such that Gram negative bacteria appears in
pink/red colour whereas Gram positive bacteria due to
crystal violet iodine complex stain doesn't get stained
with secondary stain, it remains purple/blue in colour.
This stain is less noticeable in the cells that still contain
the crystal violet-iodine complex.
 BY YASA MWELWA

Example of Gram positive bacteria is Bacillus cereus and Gram negative bacteria is
 BY YASA MWELWA
Escherichia coli.
 BY YASA MWELWA

The purple, crystal-violet stained cells are referred to as gram-

positive cells, while the red, safranin-dyed cells are gram-negative.
 BY YASA MWELWA

What purpose does Gram staining

serve?
• The main purpose of Gram staining is to
differentiate the bacteria into two broadly
classified groups - Gram positive and Gram
negative on the basis of their structure
including the cell wall content, lipid content,
response to lysozyme treatment, antibiotic
susceptibility etc.
 BY YASA MWELWA

• Gram Positive bacteria retain the primary stain

(Crystal violet) and appear purple at the end of
staining upon microscopic observation. Gram
positive cell wall does not get decolorized as it
contains lower lipid content.
• Whereas, Gram negative bacteria do not retain
the primary stain and gets decolorized on the
action ethanol because of the fact that, it acts as
lipid solubilizer. Gram negative bacteria have high
lipid content. Due to ethanol, pores are created
and primary stain gets removed. At the end of
Gram staining, gram negative cells appear pink
due to counter stain (safranin).
 BY YASA MWELWA

• Gram staining technique is to distinguish the

two types of bacteria based on the difference
in their cell wall structures. The gram-positive
bacteria retain the crystal violet dye, which is
because of their thick layer of peptidoglycan
in the cell wall.
• This process distinguishes bacteria by
identifying peptidoglycan that is found in the
cell wall of the gram-positive bacteria. A very
small layer of peptidoglycan is dissolved in
gram-negative bacteria when alcohol is added.
 BY YASA MWELWA

Difference between Gram-Positive and

Gram-Negative Bacteria.
• The cell wall of gram-positive bacteria is
composed of thick layers peptidoglycan while the
cell wall of gram-negative bacteria is composed of
thin layers of peptidoglycan.
• In the gram staining procedure, gram-positive
cells retain the purple coloured stain, gram-
negative cells do not retain the purple coloured
stain.
• Gram-positive bacteria produce exotoxins.
• Gram-negative bacteria produce endotoxins.
BY YASA MWELWA
 BY YASA MWELWA

PEPTIDOGLYCAN LAYER
DIFFERENCE
 BY YASA MWELWA

GRAM NEGATIVE BACTERIA

• Have high resistance to antibiotics than gram
positive
• Have hard protective thin outer shell. When
their cell wall is disturbed, they release
endotoxins that can make your symptoms
worse.
• They have ability to cause a lot of diseases in
humans
• Diseases caused include cholera, typhoid, etc.
 BY YASA MWELWA

GRAM POSITIVE BACTERIA

• Have a thick peptidoglycan layer (cell wall)
• Treatment used are antibiotics like
erythromycin, penicillin, cloxacillin, etc.
• Antibiotic resistance is becoming a serious
problem. New medicines are being developed
to help with this problem.
BY YASA MWELWA

END