BIOL 1017—CELL BIOLOGY

Lecture 2:
Prokaryotes & Microscopy

Dr. Sacha-Renée Todd

 In today’s lecture we will:

 Discuss the reasons for small cell

sizes
 Examine the features of prokaryotic
cells
 Review the use of microscopy to
examine cells
 Cell Size
• Even the largest creatures are made of cells

• Despite the fact that life on earth has evolved

into organisms of varying complexities, cells
themselves are very small e.g. a typical animal
cell measures ~10 microns/0.001 cm in diameter

• A pint of blood has over 5 billion specialized cells

floating in it, and you scrape off your skin or
slough off from your intestines close to 700
billion cells a day!
Why are Cells Small?
 Factors Affecting Cell Size
1.Speed of
– Intercellular Communications
– Nutrient absorption and waste expulsion
processes

 Numerous chemical reactions occur in cells.

 Substances must move from one site to
another within the cell.
 Cell Size and Speed
Small cell size is practical necessity:
 The smaller the cell:
 The shorter the distances that substances
must move; and
 The faster the movement of substances
within cells.

i.e. Shorter is Faster!

 Factors Affecting Cell Size

2. Surface Area to Volume Ratio

 As object increases in volume:
 Surface area also increases, but at much
lower rate
 Surface area to volume ratio decreases
 Cell Size
Biological significance:

Since
 Volume is proportional to:
The rate of chemical activity in the cell
&
 Surface area is proportional to:
The rates of raw material entry into, and
waste product exit from, the cell.
 Cell Size
Biological significance:
 Large cells:
 Have higher rates of chemical activity;
 Have lower rates of supplying raw
materials and removing wastes.
 Are relatively inefficient.
 To maximise efficiency, cells are small.
 Large organisms must consist of many small
cells (i.e., be multicellular).
 3. Role of Gravity?
• Recent research suggests that Gravitational
Force imposes a size limit on cells
• Gravity becomes negligible at a certain
smallness of scale, but becomes important at
a certain particle density and a cell size of
roughly ….
• 10 microns — the size limit of most animal
cells
 Implications of Cell Size for
Multicellular Organisms
Renewal
• Cells have a finite life span
• Cells arise by division, specialize, function and carry out
their roles, then age and eventually die or are lost. The
total organism remains the same throughout this process,
and (usually) has a longer time on earth than any one of its
cells
• This renewal goes on constantly throughout the life time of
a multicellular organism
• Many small cells are easier to replace than would be the
case if an organism was made up of just a few very large
cells
 Cell Classification
All cells share certain features:
1. Plasma membrane encloses cell, separating
intracellular and extracellular environments.
2. Ribosomes (sites of protein synthesis).
3. Material enclosed in the plasma membrane is
the Cytoplasm. Cytoplasm has 2 components:
• The aqueous cytosol (water with dissolved
substances).
• Insoluble suspended particles.
 Cell Classification
Living things classified into three domains:
 Archaea
 Bacteria
 Eukarya

Archaea Bacteria Eukarya

 Cell Classification
Cells are classified according to:
1. If their genes are enclosed by a nuclear
envelope.
2. If they possess membrane-bound intracellular
compartments, or organelles.

 Archaea and Bacteria do not satisfy criteria (i.e.,

are prokaryotic). Eukarya satisfy both criteria
(i.e., are eukaryotic).
 Pro = “Before” (evolutionarily earlier)
 Eu – “True” Karyon = “Nucleus” {greek}
 Prokaryotes
Prokaryotes are:
 Single-celled (unicellular) organisms.
 The earliest forms of life:
 2 billion years before eukaryotes.
 About 10 times smaller than eukaryotes:
 Often called microbes.
 Not as complex as eukaryotes:
 Lack organelles.
 DNA not separate from rest of cell, but
coiled in part of cytoplasm (nuceloid).
 Prokaryote Sociality
 Prokaryotes are unicellular, but can form
aggregate communities.
 Not truly multicellular:
 Not founded by
single founder.
 Truly multicellular
organisms originate
from single cells
Bacterial aggregate community.
(e.g., zygote).
 Features of Prokaryotes
Consider bacteria as typical prokaryote.

Nucleiod Cytoplasmic area with single

DNA molecule (haploid, with
only 1 copy of each gene).
Ribosomes Sites of protein production;
smaller than in eukaryotes.
Storage granules Cytoplasmic nutrient stores.

Plasmids Circular DNA structures; not

involved in reproduction.
 Features of Prokaryotes
From the outermost structure moving inward, bacteria have
some or all of the following:

Capsule Polysaccharide (or protein) layer in

some bacteria; protects cell when
engulfed by other organisms.
Outer membrane Additional membrane in Gram negative
bacteria; source of toxins against
immune cells.
Cell wall Composed of peptidoglycan (polymer of
polysaccharides and protein); protects
cell and gives it its shape.
Plasma Surrounds the cytoplasm; regulates flow
membrane of substances in and out of the cell.
Prokaryote Structures
Bacterial structures
 Features of Prokaryotes -
FORMS
Cell walls give prokaryotes four basic shapes:
 Cocci (sing., coccus): spherical.
 Bacilli (sing., bacillus): rod-shaped.
 Spirilla (sing., spirillum): spiral-shaped (a.k.a.,
spirochaetes).
 Vibrio: comma-shaped.

Mycoplasma are bacteria with no cell wall:

 Thus have no definite shape.
 Features of Prokaryotes –
Appendages

Bacteria may have the following appendages:

Pili Hollow, hair-like protein

(sing., pilus) structures; allow
attachment to other cells.
Flagella Long, whip-like protrusions
(sing., flagellum) that aid in cellular
locomotion; one to many
present.
Features of Prokaryotes
Bacterial appendages
 Features of Prokaryotes -
Reproduction
Most haploid prokaryotes reproduce asexually
by binary fission:
 Single DNA molecule replicates, forming two
copies.
 Plasma membrane invaginates (folds
inward) between the two DNA molecules.
 Cell wall forms between the DNA molecules.
 Original cell is divided into two identical
daughter cells.
Binary Fission in Bacteria
 Gram Stain Reaction
Method devised by Hans Christian Gram (1884)
Bacteria can be sub-divided into two classes:
 Gram positive (Gram +); and
 Gram negative (Gram -).

Gram stained mix of Staphylococcus

aureus (Gram +, purple) and Escherichia
coli (Gram -, pink).
 Gram Stain Reaction
Gram staining:
 Leaves Gram + bacteria purple and Gram –
bacteria pink.
 Depends on the cell wall:
 Gram + cells have more (up to 90%)
protective peptidoglycan in cell wall (more
infectious).
 Gram - cells have less (under 20%) protective
peptidoglycan in cell wall (less infectious).
 Cyanobacteria
 A primitive and successful group of prokaryotes (3.5
billion years old) consisting of >1500 species

 Gram-Negative bacteria that obtain energy through

photosynthesis

• Occur as individual cells, colonies or long

filamentous chains
• Lack flagella but are able to perform movement by
rotary motion or gliding over a gelatinous layer
secreted through the cell surface
 Cyanobacteria
 Are also known as blue-green algae:
 In addition to chlorophyll, contain unique
bluish photosynthetic pigments Phycobilin

 Have photosynthetic machinery called thylakoids

in invaginations of the plasma membrane that
increase surface area for photosynthetic
reactions
Cyanobacterial Diversity

© Mcgraw-Hill Companies
 Archea
• Although Archea are classified as prokaryotes along
with bacteria, they are very different!
• Archaea look like bacteria: they are tiny, single-
celled, and relatively simple with similar cytological
features
• However, they are actually more closely related
to eukaryotes
• Possess unique characteristics
• e.g. Archaea have a more stable membrane
chemistry than bacteria and eukaryotes which may
make them better able to survive in extreme
environments.
 Features of Prokaryotes
Archea v. Bacteria
Archaea and bacteria differ in:
 Bonds linking their membrane molecules.
 Archaeal cell walls are made of protein but unlike
bacteria, these lacking peptidoglycan
 Archaeal genes and protein synthesis more
eukaryotic than bacterial.
 More archaea than bacteria tend to be
extremophiles:
 i.e., thriving in physically or chemically
extreme conditions.
 Comparison of Some Properties of Bacterial, Archeal and Eukaryotic Cells
Characteristic Eubacteria Archaea Eukaryotes
Not always present
gram +ve or gram –ve murein absent
Cell wall properties (composition) (cellulose – plants;
(Peptidoglycan) (protein)
chitin – fungi)
Predominantly multicellular no no yes
Nucleus, membrane bound organelles no no yes
Circular, associated Linear, associated
Circular, few
Typical form of chromosomal DNA with histone-like with histone-like
associated proteins
proteins proteins
Ribosome size and number of proteins 70s with 55 proteins 70s with 65 proteins 80s with 78 proteins
Ribosomeal RNAs Bacterial type Archeal type Eukaryotic type
Not ester-linked
ester-linked
ester-linked (Glycerol- (Glycerol-3-
(Glycerol-3-
Cell membrane phospholipids 3-phosphate + linear phosphate +
phosphate + linear
fatty acids) branched
fatty acids)
polyisoprenoids)
Photosynthesis with chlorophyll yes no yes
Growth above 80o C yes yes no
RNA processing minimal moderate extensive
Transcription and Translation initiation Bacterial type Eukaryotic type Eukaryotic type
Nitrogen Fixation yes yes no
Chemolithotrophy yes yes no
Gas vesicles present yes yes no
 What is Microscopy
The technology of making small things visible to
the human eye
Microscopes - Instruments used to view objects too
small to be seen with the unaided eye
 Allow cellular details to be seen by:
 Increasing the apparent size of the object
(magnification).
 Making the magnified object sharp, or clear
(resolution).
 The ability to magnify and resolve an object is
based on the fundamental principles of light
 Size, Scale & Units of Measurement

Meter Centimeter Millimeter Micrometer Nanometer Angstrom Picometer

100 m 10-2 m 10-3 m 10-6 m 10-9 m 10-10 m 10-12 m
1m 0.01 m 0.001 m 0.000001 m 0.000000001 m 0.0000000001 m 0.000000000001 m
1/1,000,000,000 1/10,000,000,000 1/1,000,000,000,000
1/100 m 1/1,000 m 1/1,000,000 m
m m m

one one one one ten one

hundredth thousandth millionth of billionth of billionth of a trillionth of a
of a meter of a meter a meter a meter meter meter
• Resolution:- The smallest distance two objects
can be apart and still be seen as two objects

The distance
between two
adjacent
crests/troughs
of any wave is
a wavelength,
λ

• Light must pass between two objects for them

to be seen as separate.
• If the wavelength of light is too long to pass
between them, they will appear as one
An analogy for the effect of wavelength on resolution
Smaller objects (corresponding to shorter wavelengths)
can more easily pass between the arms of the letter E,
defining it more clearly and producing a sharper image
© 2005, John Wiley and Sons Publishers
 Increasing Wavelength

Increasing Energy Level and Resolving Power

The electromagnetic spectrum

Only a narrow range of wavelengths are used in microscopy
 Microscopy
Two basic types of microscopes:
 Light microscopes.
 Electron microscopes.

Light microscope Electron microscope

 Light Microscopy
• The use of any kind of microscope that uses a beam
of visible light to make specimens observable
• Light microscopes use a system of lenses to magnify
an image.
• The power of a light microscope is limited by the
wavelength of visible light, about 550 nm. These
cannot resolve distances less than 220 nm.
• There are microscopes which use ultraviolet light
(instead of visible light). The wavelength of UV light is
between 100-400 nm. These microscopes can resolve
distances as small as 110 nm.
 Comparison of Light (Optical) Microscopy Techniques
Bright-field Light passes directly Unless natural pigments are
microscopy: through the cells present, there is little contrast
and little detail .

Stained bright- Stains are used that Stains may bind to specific cell
field enhance contrast, materials.
microscopy: revealing details not
otherwise visible

Fluorescence Uses UV light instead of The molecules emit different

microscopy: white light to stimulate wavelengths, often of brilliant
molecules within the colours.
specimen or dye
molecules
Dark-field Utilizes a special Light reflects off the specimen
microscopy: condenser at an angle rather than pass
through. Thus, the field around
the specimen is dark.
Phase -contrast Utilizes a special Contrast is increased by
microscopy: condenser emphasising differences in the
refractive index (capacity to
bend light) between cell
structures. i.e. Enhances light
and dark regions in the cell .
 Electron Microscopy
• Electron microscopes shoot a high-voltage beam of
electrons (instead of light) onto or through an object,
which deflects and absorbs some of the electrons.
• Electromagnets are used (rather than glass lenses) to
focus the beam
• Resolution is still limited by the wavelength of the
electron beam, but this wavelength is much smaller
than that of visible light.
• Electrons behave both as particles and as waves.
Their wavelength is about 0.005 nm which allows
resolution of distances as small as 0.2 nm.
ILLUSTRATIONS contrast the architectural structure of,
and images produced by light and electron microscopes

ILLUSTRATIONS contrast the structure of an intestinal

cell as known from light vs. electron microscopy (left
and right) (From Sheldon, 1958)
 Comparison of Electron Microscopy Techniques

Transmission Very thin Magnets focus electron

electron sections of a beams on the object.
microscopy specimen are Electrons that pass
(TEM): used, revealing through are detected on
the internal a fluorescent screen.
structure of Things that absorb
cells electrons appear darker.

Scanning Specimen is Directs electrons to the

electron coated with a sample’s surface.
microscopy metal. The Excited sample then
(SEM): electron bean is emits other electrons.
scanned or These produce an image
swept over this of the object’s three-
coating to form dimensional surface on a
a 3-dimensional screen.
image
 Characteristics of Microscopes
Characteristic Compound Light Microscope Transmission E. Microscope Scanning E. Microscope

Resolution (Average) 500 nm 10 nm 2 nm (3-D)

Resolution (Special) 100 nm 0.5 nm 0.2 nm (3-D)

Magnifying Power up to 1,500X up to 5,000,000X ~ 100,000X
Depth of Field poor moderate high
Type of Objects living or non-living non-living non-living

Preparation Technique usually simple skilled easy

Preparation Thickness rather thick very thin variable

Specimen Mounting glass slides thin films on copper grids aluminum stubs

Field of View large enough limited large

Source of Radiation visible light electrons electrons
Medium air vacuum vacuum

Nature of Lenses glass electrostatic + em. lenses electrostatic + em. lenses

current in the objective lens current in the objective lens

Focusing mechanical
coil coil
current in the projector lens current in the projector lens
Magnification Adjustments changing objectives
coil coil

Specimen Contrast by light absorption by electron scattering by electron scattering

Microscopy: Resolution