Microbiology: Microscopy & Cell Structure
Chapter
2: How We See the Invisible World
Chapter 3: The Cell
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I. Principles of light
microscopy-The bright-field microscope
A. In light microscopy, visible light passes
through the specimen.
1. The most type common
microscope is the _________________
_________________________________.
a. The specimen should appear dark against a
bright, white
background.
2. We use
________________________ microscopes
– the total
magnification is the product of the magnifications of the
objective
lens and the ocular lens.
3. The usefulness of a
microscope depends on its resolving
power.
a. ________________________ -
the ability of a
microscope to show separate distinct
images of two objects
close together.
b.
Resolution is affected by the amount and physical
properties
of the visible light traveling through the
microscope.
4. Lack of contrast is a
problem when viewing unstained bacteria.
a. Unstained bacteria are typically colorless
and nearly
disappear against
a white background.
B. Light microscopes that enhance contrast
1. The
________________________ microscope
has optical
devices that amplify differences in refraction.
2. The
________________________ microscope
combines two
light beams that pass through the specimen separately, causing the
specimen to appear as a three-dimensional image.
3. The
________________________ microscope
directs light
toward a specimen at an angle.
C. The fluorescent microscope
1. The
________________________ microscope
is used to
observe cells that have been stained with fluorescent dyes.
2. The fluorescent
microscope projects ultraviolet light through the
objective lens and onto the specimen.
D. The confocal scanning laser microscope
1. The confocal scanning
laser microscope is used to construct
a three-dimensional image of a thick structure and to provide
detailed sectional views of the interior of an intact cell.
2. A laser beam to scans
across and through a specimen; a
computer uses that data to construct the image.
E.
Electron Microscopes
1. Electron microscopes use
electromagnetic lenses, electrons,
and phosphorus screens to produce a magnified image.
2. ________________________________________________
(TEM) transmit electrons through a specimen that has been
prepared by thin-sectioning, freeze fracturing, or freeze etching.
3. Scanning electron microscopes scan a beam of electrons
back and forth over the surface of a specimen, producing a three-
dimensional effect.
F. Scanning Probe Microscopes
1. Scanning probe microscopes map the bumps and valleys of a
surface on an atomic scale.
II. DYES AND STAINING –
remember that most microbes are clear & therefore
hard to see
A. ________________________________ – stain some components on
the
slide one color and other components a contrasting color. You can tell
the
difference between the specimens simply by looking at them.
1. The
________________________ is by far the most widely
used procedure for staining bacteria; ________________________
bacteria stain purple and ________________________ bacteria
stain pink.
a. ________________________ – crystal violet
(everything
is purple)
b. Rinse
c. ________________________ – Gram’s iodine
d. rinse
e. ________________________ – 95% alcohol + 5%
acetone
1) Gm
+ bacteria retain purple color, Gm – bacteria
are
now colorless
f. ________________________ – Safranin – stains
everything pink
1) Gm
+ bacteria still appear purple, Gm – bacteria
appear
pink
2. The
________________________ stain is
used to stain
organisms such as ________________________, which do not
take up
stains readily; acid-fast
organisms stain pink and all other
organisms stain blue.
a. 1˚
stain – ________________________ – stains
everything red
1) the slide must be heated over boiling water,
or you
must use more stain & allow
more time
b.
rinse
c.
flood with ________________________ – only the acid-
fast cells hold red color
1) the cell wall has a greater
concentration of waxy
lipid
d. ________________________
– counterstain
e.
Used to diagnose ________________________ &
leprosy (now called ________________________)
B. Special stains to observe cell structures
1. The capsule stain
is an example of a
________________________
stain; it colors the background,
allowing the capsule
to stand out as a halo around an organism.
a. ________________________ – viscous layer
around cell
sometimes
correlated with virulence
2. The spore stain
uses heat to facilitate the staining of
________________________.
3. The flagella stain employs a mordant
that enables the stain to
adhere
to and coat the otherwise thin flagella.
C. Fluorescent dyes and tags
1. Some fluorescent dyes
bind compounds that characterize all
cells, others bind to compounds specific to only certain cell
types.
2. ________________________
is used to tag a specific protein of
interest with a fluorescent compound.
III. MORPHOLOGY OF
PROKARYOTIC CELLS
A. The ________________________ of
bacteria (what you might call the
shape)
1. Most common prokaryotes
are…
a. ________________________
– spheres
b.
________________________
– rods
2. Other shapes include …
a.
________________________ – ovals
b. ________________________ – comma-shaped
c.
________________________
– loose spring
d. ________________________ – tight spring
3.
________________________
bacteria have variable shapes.
4. Aquatic bacteria come in
a variety of unique shapes.
B. The ________________________ of bacteria (what you might call the
groupings)
1. Cells adhering to one
another following division form a
characteristic arrangement that depends on the plane in which the
bacteria divide.
2. Typical cell
arrangements include …
a. chains of varying
lengths
1) 2 together are
________________________
2) 3 or more together are
______________________
b. groupings that look like a grape cluster are ____________
c. groupings of four are ________________________.
C. Multicellular associations
1. Some types of bacteria,
such as myxobacteria, typically live in
associations containing multiple cells.
2. Cells within
________________________ often alter their
activities when a critical number of cells are present
IV. THE
STRUCTURE OF THE PROKARYOTIC CELL
A. The ________________________________________________
1. Why is it important?
a. It is the gatekeeper to substances that enter
and exit a
cell.
b. It maintains cellular integrity.
c. It separates the interior environment from
the exterior and
regulates
molecule traffic flow.
2. The current model of plasma membrane
structure is the ______
________________________________________________.
a. It is only 8-millionths of a millimeter thick
b. Composed of a bi-layer of ________________________
with
partially or wholly embedded
________________________ interspersed throughout.
1) Phospholipid molecules have their
water-soluble
(phosphate) ends toward
the outsides and fat-soluble
(lipid) portions toward
the inside of the membrane.
c.
The layer is liquid, providing flexibility; embedded
cholesterols decrease this fluidity.
d.
Some of the embedded proteins function to transport
molecules across the plasma
membrane.
e.
Some proteins provide a mechanism by which
cells can
sense and adjust
to their surroundings.
3. Cytoplasmic membranes are ________________________
________________________.
a. Allows
some substances to pass freely (passive transport)
b. Actively moves
some substances either out of or into the
cell (active
transport)
c. It
inhibits the movement of other substances
d. It is
extremely important in maintaining cellular
________________________
4. _______________________________-
depends on kinetic
energy of molecules
and/or pressure gradients (things will move
from high to low
concentration without the cell expending energy)
a. ________________________
- Small, fat-soluble,
uncharged
(nonpolar) molecules (e.g. oxygen & carbon
dioxide) can
flow freely through the phospholipids from high
to low
concentration until they reach equilibrium.
b. ________________________
- movement of water
across a
membrane, along a concentration gradient
1) Water always moves from high to low
concentration,
across the plasma membrane.
2) As the solute concentration increases, the
water
concentration
decreases.
3) _____________________ solutions – “low salt”
a) Water goes into the cell
b) Animal cells would explode in this solution
c) The inflow of water
into the cell exerts more
osmotic
pressure on the cytoplasmic
membrane
than it can generally withstand;
however,
the rigid cell wall can withstand the
pressure.
4) _____________________ solutions – “high salt”
b) Water exits the cell
5) ____________________ solutions – “same salt”
c) Water has no net movement
c. ________________________________
– some
molecules
are too big (e.g. sugar), or are polar (e.g.
electrolytes),
and can’t squeeze between the phospholipids.
1) Transport proteins help these molecules
across
the
membrane.
2) These molecules still travel from high to low
concentration
(no ATP is required).
5. ______________________________
– sometimes cells have to
move molecules AGAINST
the concentration gradient (moving from
low to high
concentration)
a. Members of the major
facilitator superfamily (which
includes symporters, antiporters and uniporters) use the
proton motive force for energy.
b. ABC transport systems
require ATP for energy.
c. Group translocation
chemically modifies a molecule
during its passage through the cytoplasmic membrane.
6. Secretion
a. The general secretory pathway is the primary
mechanism used to secrete proteins.
b. The presence of a
characteristic signal sequence targets
proteins for secretion.
7. The role of the
cytoplasmic membrane in energy generation (i.e.
production of ________________________)
a. The electron transport chain within the membrane
expels protons, generating an electrochemical gradient,
which contains a form of energy called proton motive force.
b. We will revisit this
topic.
8. The membrane lipids of the archaea are
distinctly different from
those
of bacteria
V. CELL WALL of Prokaryotes
A. The chemistry of peptidoglycan
1. ________________________
is a macromolecule found only in
the bacteria and provides rigidity to the cell wall.
2. Peptidoglycan is
composed of glycan strands, which
are
alternating subunits of ________________________ (
________________________
(NAG), interconnected via the
tetrapeptide chains on
B. The Gram-positive cell wall
1. The Gram-positive cell wall contains a relatively ____________
layer of peptidoglycan.
2. Teichoic acids and lipoteichoic acids
stick out of the
peptidoglycan molecule.
C. The Gram-negative cell wall
1. The Gram-negative cell wall has a relatively _________ layer of
peptidoglycan sandwiched between the cytoplasmic membrane and
an outer membrane.
2. ________________________
contains a variety of proteins
including those involved in nutrient degradation and transport.
3. The outer membrane
contains lipopolysaccharide.
a. The ________________________
portion of the
lipopolysaccharide molecule is toxic, which is why LPS is
called ________________________ .
4.
________________________
form small channels that permit
small molecules to pass through the outer membrane.
D. Antibacterial compounds that target
peptidoglycan.
1. ________________________
binds to proteins involved in cell
wall synthesis and, subsequently, prevents the cross-linking of
adjacent glycan chains.
a. Only useful against Gram-positive bacteria
2. ________________________
breaks the bond that links
alternating NAG and
integrity of the glycan chain.
E. Differences in cell wall composition and Gram
stain
1. The Gram-positive, but
not the Gram-negative, cell wall retains
the crystal violet-iodine dye complex even when subjected to the
trauma of acetone-alcohol treatment.
F. Characteristics of bacteria that lack a cell
wall.
1. Because
________________________ do not have a cell wall,
they are extremely variable in shape and are not effected by
lysozyme or penicillin.
G. Cell walls
of the Domain Archaea
1. Archaea have a greater
variety of cell wall types than do the
Bacteria.
VI. Surface
Layers External to the Cell Wall of Prokaryotes
A. Glycocalyx
1. A ________________________ is a
distinct and gelatinous
layer
2. A ________________________ is diffuse and
irregular.
3. Both are usually made of polysaccharide.
4. Capsules and slime layers enable bacteria to
adhere to
surfaces.
5. Some capsules allow disease-causing
microorganisms to thwart
the
innate defense system.
B. Sheaths
1. A ________________________ is a tube that
holds a linear
chain
of cells; it is thought to protect the enclosed organisms from
disruption
VII.
Appendages of Porkaryotes
A. Flagella – rotate like a propeller to produce movement
1. The flagellum is a long protein
structure, composed of a
________________________,
a ________________________,
and
a ________________________, that is responsible for most
types
of bacterial motility.
a. ________________________
(flagella distributed widely
across surface) vs. ________________________ (flagella at
one end of bacteria)
2. ________________________ is the directed
movement toward
an
________________________ or away from a
________________________;
bacteria do this by adjusting the
frequencies
of their ________________________ and
________________________.
3. ________________________,
________________________
and
________________________ are directed movements toward
light,
oxygen and a magnetic field, respectively.
B. ________________________ – shorter &
thinner than flagella
1.
Many types of pili (fimbriae) enable attachment of cells to specific
surfaces
by adhesion.
2. Some pili play a role in
specific types of motility,
________________________ (short jerking) and
________________________
(smooth movement), which both
occur on solid surfaces and require cell-to-cell contact.
3. ________________________ are involved in conjugation,
which
enables
DNA to be transferred from one cell to another.
VIII. INTERNAL STRUCTURES
of Prokaryotes
A. Chromosome
1. The chromosome of
prokaryotes resides in the
________________________ rather than within a membraned
nucleus.
2. The typical chromosome
is a single, circular, double-stranded
________________________ that contains all the genetic
information required by a cell.
a. It is tightly packed into 10% of the cell
volume –
supercoiled
B. Plasmids
1. ________________________ are circular,
double-stranded
DNA molecules that typically encode genetic information that may
be advantageous, but not required by the cell.
a. They carry a few to several hundred genes
b. Some plasmids carry ________________________
&
________________________
resistant enzymes
2. Populations of cells can
gain and lose plasmids, depending on
the relative advantages.
a. Information can be advantageous if the cell
is exposed to
that antibiotic,
for example.
b. Cost – _______________________________________
________________________________________________
1) In
the absence of that antibiotic, cells without this
plasmid
will replicate faster & thereby out-compete
the
mutant, resistant strain
2) Where
would there be a great breeding ground for
antibiotic
resistant bacteria?
3)
How could we break the vicious cycle of antibiotic
resistance?
c. Cultures can be “cured” of a plasmid
C. ________________________ are the workbenches
upon which
proteins
are constructed
1.
________________________ are long chains of
________________________ that take
on complex shapes.
2.
Proteins may be structural (like membranes) or functional (like
enzymes)
3. The
_________________________________ is composed of a
50S and a 30S subunit.
a. S is the relative size & density of
ribosomes & their
subunits
4. Eukaryotic cells are
80S.
5. Some antibiotics target
70S & inhibit protein synthesis in
bacteria, leaving the patient unaffected.
D. ________________________
1. Storage granules are dense accumulations of high molecular
weight polymers, which are synthesized from a nutrient that a cell
has in relative excess.
a. If N & P are absent, the cell can’t
replicate, but it can
store C and energy as glycogen if they are available.
b. This lets them prepare
to divide & then wait for N
& P to become available.
E. ________________________
1. Gas vesicles are gas-permeable, water-impermeable rigid
structures provide buoyancy to aquatic cells, enabling the cell to
float or sink to an ideal position in the water column.
a. Increase # of vesicles, elevate position in
water column
F. ________________________
1. Endospores are a dormant stage produced by members of
________________________ and ________________________;
they can germinate to become a vegetative cell.
2. Endospores are
extraordinarily resistant to conditions such as
heat, desiccation, toxic chemicals, and UV irradiation.
a.
________________________,
________________________, _______________________
and ________________________ are diseases caused by
endospore-formers.
3. ________________________
is an eight-hour process initiated
when cells are grown in nutrient-limiting conditions.
4. ________________________
is the process by which an
endospore leaves its dormant state.
a. NOT reproduction.
b. 1 vegetative cell à 1 endospore à 1 vegetative cell
c. Reproduction is 1 vegetative cell à 2 daughter vegetative
cells
IX.
EUKARYOTIC CELL STRUCTURE
A. The plasma membrane - the outermost membrane, containing the
nucleus and the cytoplasm – the organelles and the fluid they are
suspended in.
1. What we learned of the bacterial cytoplasmic
membrane is true
of this membrane as
well.
2. This is a phospholipids bilayer with wholly
and partially
embedded
proteins.
a. Proteins in the membrane
are involved in transport,
structural integrity and signaling.
b. Sterols provide strength to the fluid
membrane.
3. This membrane is selectively (or differentially) permeable
a. Small, nonpolar molecules diffuse through the
membrane
b. Water freely flows across the membrane by osmosis
c. Some larger or charged molecules are carried
across
through
protein channels by facilitated transport.
d. Some molecules are moved against
concentration
gradients
through the use of ATP by active
transport.
1) Carriers involved in
active transport include
members of the major facilitator superfamily and ABC
transporters.
2) e.g. K+ levels
inside cells are often 20-50 times
higher than outside levels, while Na+ levels may be 10
times higher outside than inside.
e. This control of what enters and leaves the
cell is how the
cell
maintains homeostasis.
4. Vesicular
transport – the plasma membrane surrounds and
moves
large amounts of material in an enclosed vesicle
a. These movements always requires
ATP.
b. Endocytosis
encloses a particle in a vesicle that is
engulfed. (Material moves into the cell.)
c. Phagocytosis
literally means “cell eating.”
1) An
area of the plasma membrane forms a pocket to
engulf
material.
2) The
membrane-enclosed vesicle detaches from the
cell
surface for internal digestion.
3) This produces a food vacuole.
d. Pinocytosis:
“cell drinking”
1)
Movement of dissolved particles into the cell
e. Exocytosis
- the reverse of endocytosis, it moves
materials
out of the cell.
1) Expels indigestible residues
2) Secretes hormones and transport substances.
B. The Nucleus
(the control center of the cell)
1.
The nucleus is surrounded by a double-layered nuclear
envelope.
a. This membrane has large pores to let
molecules in and
out.
b. The nuclear envelope is continuous with the
endoplasmic
reticulum.
2.
Inside the nuclear envelope is the chromatin
network.
a. Chromatin
is a threadlike material that coils into
chromosomes just before
cell division occurs; it contains the
DNA
b. DNA
in the nucleus provides information needed to make
proteins, grow, differentiate, and carry
on other activities
c.
DNA also stores hereditary information
3. Nucleolus
– darkly-staining body in the nucleus
a.
It produces the ribosomes.
C.
Endoplasmic Reticulum (ER) (the
manufacturing plant of the cell)
1. A system of interlinked
double-membraned channels subdividing
the cytoplasm
2.
Primary site of membrane synthesis in the cell
3.
ER comes in two varieties: rough
& smooth
a. Rough
ER is rough because it is studded with
ribosomes
1) Ribosomes
–the workbenches upon which
proteins are built.
a)
Ribosomes have no bounding membranes
and therefore aren’t considered to be
organelles by many.
b)
The 80S eukaryotic ribosome is composed
of 60S and 40S subunits
2)
Rough ER synthesizes, stores, and secretes
proteins.
1) Proteins may be
structural & form
organelles
or membranes.
2) Proteins may be functional (e.g. enzymes)
3)
Products of the rough ER are transported to the
Golgi complex for storage or activation.
b. Smooth ER has few if any ribosomes
1) Smooth ER synthesizes lipids and
phospholipids.
D. Golgi complex (the warehouse of the cell)
1. Disc-shaped, often
branching hollow tubules just outside the ER
2. It receives products from the ER, and does
one of three things…
a. Stores the product for later use by the cell
b. Modify the product
1) e.g. It may modify carbohydrates attached to
proteins
to activate an enzyme
c. Ship the product for use elsewhere in the
cell
1) Collects product in small vesicles that are
pinched
off
from the margins.
3. It produces lysosomes.
a. Lysosomes are membrane-bound vesicles
that contain
digestive enzymes.
b.
They help digest foreign material or engulfed bacteria by
fusing with a food vacuole produced by
phagocytosis.
c.
They destroy injured or diseased cells.
4. Peroxisomes are the
organelles in which oxygen is used
to oxidize certain
substances.
E. Mitochondria (the Powerhouses of the cell)
1. The energy (E) stored in
sugar is released by the process of
cellular respiration here.
a. The sugar is broken down into water and
carbon dioxide,
releasing
energy.
b. That energy is stored in a form the cell can
then use to do
work – ATP (adenosine triphosphate).
2. Mitochondria are shaped
like cucumbers, rods, or balls, they
move throughout the cell
and accumulate where energy is needed.
3. Bounded by 2 membranes, the inner
membrane forming platelike
folds called cristae which increase the surface area for enzymes to
work on.
4.
Mitochondria are self-replicating and have their own DNA.
F.
Chloroplasts – the green
organelles in plants and algae where
photosynthesis takes place.
1. They contain chlorophyll
2. They utilize the energy of light to make
sugar.
G. Cytoskeleton - a network of filaments and tubules that maintain
support and form.
1. In many cells, they provide locomotion and
translocation of
organelles.
2.
Microtubules are the thickest of the cytoskeleton structures and
are long hollow cylinders
3. Microfilaments
allow the cytoplasm to move and are composed
of actin.
a. The flow of
cytoplasm is known as cyclosis
or
cytoplasmic streaming.
4. Intermediate
filaments strengthen the cell mechanically.