Microscopy and Cell structure
• Microscope- tool used to see objects too small
to be seen by the naked eye. Types of
microscopes used in the study of
microorganisms include the light microscope,
dark field microscope, phase contrast
microscope, confocal microscope, interference
microscope, fluorescence microscope, electron
microscope, and atomic force microscope.
Terms to know
• Bright field microscopy- lights rays are used to
evenly illuminate the field of view.
• Dark field microscopy- light is directed
towards the specimen at an angle. This makes
it possible for the unstained specimen to
appear more visible against a dark background.
• Contrast- basically it is the number of visible
shades in a specimen.
• Microscopes that increase contrast include,
phase contrast microscope, interference
microscope, dark field microscope,
fluorescence, and confocal microscope.
1-Light microscope- uses a beam of light to
create an enlarged image of the specimen. The
light microscope can either use a mirror or a
light bulb to pass light through the specimen.
You can magnify a specimen up to 1000x with
a good light microscope
Parts of the microscope
A- body tube- connects the eyepiece with the
B-eye piece- the lens the observer looks through.
C-rotating nosepiece- holds the objectives and
allows you to switch from one to the other.
D-objective lens- the lens found on the nosepiece
that magnifies the image. Most microscopes
contain three; the low power, high power, and
the oil immersion lens.
• *To determine the total magnification power,
multiply the power of the eyepiece with the
power of the objective lens. For example; if
the eyepiece is 10x and you are using the high
power, which is 40x, then the total magnifying
power is 400x. So, it means you are looking at
the object 400x closer.
E-coarse adjustment knob-allows you to move the stage
closer to the objective lens and focus the specimen.
When you first place a slide in the scope, you should
always start with low power and focus with the
F-fine adjustment knob-allows you to move the stage
very slowly and finely focus the specimen under high
power. Keep in mind that when you move to high
power; the amount of light passing through will
diminish. You might need to adjust the diaphragm.
G-stage- broad flat platform where you place the
H-stage clips- hold the slide in place.
I-diaphragm- a circular flat wheel underneath the
stage that allows you to control the amount of
light passing through.
J-arm- used to carry the microscope and for
K-light source- provides the light needed to
create an enlarged image of specimen. The
light source is either a mirror or a light bulb.
Resolution- the ability of a microscope to
separate and show two points that are very
Resolution depends on the type of lens used,
wavelength of the light, magnification, and
Resolution can be expressed as
d= .5 (wavelength)/ NA
d= distance between the two points
NA = numerical aperture, a mathematical
expression that describes how well the
condenser directs light rays through the
To increase resolution
• The maximum resolving power of the best
light microscope is .2 um due to the
wavelength of light.
• When using the 100x objective lens, one must
use immersion oil to maximize resolution. This
is because of the difference in the refractive
index of air and glass. As light rays pass from
one medium to another, light rays will bend if
there is a difference in the refractive index.
• Refractive index is the velocity of light as it
travels through a medium.
Electron microscope- uses a beam of electrons
to create an enlarged image of the specimen.
An electron microscope can magnify a
specimen up to 250,000x closer. The
wavelength used is less than the wavelength of
light, thus the resolution is greater.
• TEM- transmission electron microscope allows
one to see fine detail structures in side the cell.
• SEM- scanning electron microscope allows
one to see the surface details of cells like the
3-Phase contrast microscope- allows you
to see unstained cells by altering the
background of the cell. This microscope
has a device that allows it to amplifies
differences in refractive index to create a
Projects ultra violet light causing fluorescent
molecules in the specimen to emit a longer
5- Confocal microscope
Mirrors are used to scan a laser beam across
successive regions and planes of a specimen. A
computer program constructs a 3D image.
6- Interference microscope causes the
specimen to appear as a 3D image. The
most common one is the Nomaski
differential interference microscope. This
microscope has a special device that
separates light going through a specimen
into 2 beams and then recombines them.
The light rays are out of phase when they
recombine, yielding a 3D image.
7- Dark field microscope
this type of light microscope has a special
device that directs light at an angle so that only
light scattered by the specimen enters the
objective so one sees the specimen against a
8- Atomic force microscope
This very powerful microscope produces a
very detailed image of the surface of an
specimen by using a very sharp probe (stylus)
to move across the surface and “feel” the
bumps and valleys of the atoms of the surface.
• Use of either a basic or acidic dye to color
certain cell parts and make it more visible to
the naked eye. Three types of staining
techniques; simple, differential and special
stains for the flagella, spores, and capsule.
• Basic dyes carry a positive charge. They stain
the negative parts of a cell like proteins and
nucleic acids. Examples include methylene
blue, crystal violet, safranin, malachite green.
• Acidic dyes carry a negative charge. They are
usually used to stain the background to
observe colorless cells.
• Neutral no charge
• Staining allows you to see parts of the cell you
would not ordinarily see. Cells are clear! By
adding a stain you are basically coloring
certain parts of the cell that take in the dye.
Three basic shapes; spherical called coccus,
cylindrical called bacillus and spiral. There
coccobacillus- short rod
vibrio- a short curve rod
spirochete- a long helical cell with a flexible cell
wall and unique mode of utility
Pleomorphic- bacteria that vary their shape
1- fruiting body
Structure of prokaryote cell
1-Flagella provides motility. The flagella is
made up of three basic parts, filament, hook
and basal body.
2-Pili proteins that enable the bacteruim to
adhere to surfaces. Fimbriae allow bacteria to
adhere to surfaces and sex pili allow DNA
transfer between bacteria.
3-The capsule a viscous and gelatinous layer that
surrounds bacteria. It enables bacteria to
adhere to certain surfaces and allows
organisms to avoid innate defense systems and
cause diseases. Ex, Streptococcus pneumoniae.
4- slime layer- gel like layer that is diffuse and
irregular. This layer is composed of
polysaccharides and enables the bacteria to
adhere to surfaces and grow as biofilm.
Ex; Streptococcus mutans grows as biofilm on
your teeth to form dental plaque.
5- The cell wall a rigid covering consisting of
peptidoglycan that gives the bacterium its
shape and protection.
• The type of cell wall distinguishes between 2
types of bacteria; gram negative and gram
• Peptidoglycan is a macromolecule found only
• The basic structure of peptidoglycan is an
alternating series of 2 major subunits, N-
acetylmuramic acid (NAM) and N-
acetylglucosamine (NAG). These subunits are
covalently bonded to each other to form a
• Attached to each NAM molecule is a string
of 4 amino acids, a tetrapeptide chain.
Cross linkages can form between adjacent
chains thus joining adjacent glycan chains.
• In gram negative bacteria these tetrapeptides
are joined directly.
• In gram positive bacteria they are usually
joined indirectly by a peptide interbridge.
• In gram positive bacteria the peptidoglycan
layer is thick.
• In gram negative bacteria the peptidoglycan
layer is thinner.
• In gram positive bacteria there are polymers of
teichoic acid present. These teichoic acid
polymers are covalently linked to the NAM
molecules of the glycan chain.
• These polymers consists of chemically
modified ribose or glycerol sugars connected
by phosphates. Sugars and D- alanine may be
attached to these polymers providing antigenic
• Teichoic acid provides rigidity to the cell wall.
• In gram negative bacteria there is an outer
membrane outside the peptidoglycan. It is a
unique lipid bilayer.
• The outer membrane is unlike any other
membrane. The outside leaflet consists of
Lipopolysaccharides instead of phospholipids.
• The outer membrane is sometimes called the LPS or
• The outer membrane is joined to peptidogylcan by
means of lipoproteins
• Two parts are importance for medical reasons;
a- Lipid A is the portion that anchors the LPS in the
lipid bilayer. It plays a role in the immune system.
b- O specific polysaccharide is a chain of sugar
molecules opposite the Lipid A. Allows for
• In gram negative bacteria, it is the space
between the inner and outer membrane.
6- Bacterial chromosome is usually circular
double stranded molecule located in a region
called the nucleoid
7- Plasmids are small, supercoiled, circular
double stranded pieces of DNA that contain
8- Endospore is a type of dormant cell that
resists harsh conditions.
9- Cytoskeleton proteins that provide support.
10- Gas vesicles provide buoyancy
11- granules are accumulations of substances produced
• Examples are glycogen, poly beta hydroxybutyrate,
• Volutin- a storage form of phosphate. They stain red
with methylene blue, sometimes called
metachromatic granules. Role is unclear. Thought to
be involved in energy storage and pH balance inside
12- ribosomes involved in protein production
1- Plasma membrane consisting of asymmetrical
The cell membrane consists of proteins and
Internal protein parts
2- Cilia/flagella are protein structures that
consists of microtubules in a 9 +2
arrangement. Cilia and flagella function in
3- cytoskeleton consists of proteins such as
microtubules, actin filaments and intermediate
filaments that function in cell structure/support
and act as a molecular monorail.
4- ribosomes are involved in protein production.
5- Chloroplast- double membrane bound
organelle involved in photosynthesis
6- Endoplasmic reticulum is a system of canals
involved in the production of macromolecules
destined to be secreted to other organelles or
outside. Two types, smooth and rough.
7- Golgi Apparatus is a system of flat
membranes involved in the modification of
material made in the ER. The vesicles are
coated with special carbohydrates and
phosphate groups that signal the vesicles to
their specific location.
9- Nucleus is the control center of the cell.
Contains the genetic material and is
surrounded by a nuclear envelope.
10- Mitochondrium is the site of cellular
12- peroxisomes are organelles us to oxidize
substances, breaking down lipids, and
detoxifying certain chemicals.