Leccture 3 Flashcards
(49 cards)
1
Q
Fundamentals of Light Microscopy
A
*Bright field microscopy most commonly used
*Typically employs compound microscope
*Common components:
*Light source
*Condenser lens
*Stage
*Objective lens
*Ocular lens
*Focusing knob
2
Q
Fundamentals of Light Microscopy
A
- Light passes through condenser lens, focused on specimen
- Light passing through specimen passes through objective lens,
parallel light rays now diverge - Divergent light rays pass through objective lens, further divergence
occurs - Divergence of once parallel light rays results in
magnification – increase in size of specimen - Total magnification – product of magnifying
power of objective and ocular lens
3
Q
Magnification
A
- Not the rate limiting factor (by far!)
- Magnification can be infinite
- At some point, no more information can be gathered
- Have reached the limiting factor of microscopy - RESOLUTION
4
Q
Resolution
A
- Minimum distance at which two objects can be distinguished
- Several factors affect resolution
- Wavelength of light used
- Refractive index of medium between lens and specimen
- Distance between lens and specimen
- Contrast
l = wavelength of light
h = refractive index
q = angle between most
divergent light ray gathered by
lens and the center of the lens
5
Q
Wavelength of Light Used
A
- Different colors of light have different wavelengths
*The shorter the wavelength, the higher the resolution - Many light microscopes use filters to select color of light
- What color of light in the visible spectrum would
provide the highest resolution?
6
Q
Refractive Index of Medium
Between Specimen and Lens
A
- Refractive index - ability to bend light
- Glass from the slide bends light more than air
- Light is bent away from lens as it passes through specimen
- Immersion oil has similar refractive index as glass
- More light is gathered by the lens, more information
7
Q
The Distance Between the
Lens and the Specimen
A
q = angle between most
divergent light ray
gathered by lens and the
center of the lens
The closer the lens to the specimen - the greater the value of q
8
Q
The Distance Between the
Lens and the Specimen cont
A
- Object is to obtain information
- In microscopy, light is information
- The further the lens from the
sample, the more light is lost - The closer the lense, more light
gathered
*More light means more information
and higher resolution
9
Q
Bright-Field Microscopy
A
- Condenser creates a
bright white
background against
which to see
specimens - Cells and organelles
within them are often
times transparent - Need a way to produce
contrast
10
Q
Preparing Cells for Staining
A
- Specimen must be very thin, thin sectioning for tissues, smears for
bacteria - Specimen must dry, allows for fixation
- Fixation attaches specimen to slide, preserves structure, multiple
ways to accomplish, heat or methanol common fixation techniques - Most simple stains – basic stains, (+) charge, binds to (-) charged
cell, electrostatic interaction - Net result, increases contrast
11
Q
Differential Staining – the Gram Stain
A
- Specimen prepared as for simple staining, 1 stain added – crystal violet
- Mordant added – iodine, causes crystal violet to form large aggregates – CVI
- Cells decolorized with alcohol
- Gm (+) cells retain CVI, Gm (-) cells decolorized
- 2 stain (counter stain) allows visualization of Gm (-) cells, safranin
- Gm (+) cells appear purple, Gm (-) cells appear red, provides information on structure
12
Q
Structure of the Gm (+) Cell Wall
A
- Outermost layer – peptidoglycan, composed of sugar and protein
- Alternating N-acetylglucosamine and N-acetylmuramic acid joined in
long chains by b-1,4 linkage - Form long chains that surround the cell
- Chains linked together by peptide crosslink, type of peptide depends
on species
13
Q
Structure of the Gm (+) Cell Wall cont
A
- Teichoic acids embedded in peptidoglycan layer
- Polyalchols composed of repeating residues of glycerol
phosphate or ribotol phosphate - Usually have sugars and D-Alanine attached to hydroxyl
groups of alcohol, always have phosphate ester group
attached - Negative charge allows binding of divalent cations (Ca++ and
Mg++) - Believed to have role in uptake of ions bound
14
Q
Structure of the Gm (-) Cell Wall
A
- Outermost structure is the outer membrane – lipid bilayer
- External surface of membrane contains unusual lipid – lipopolysaccharide – bacterial
endotoxin - Two components – polysaccharide chain and lipid A
- 2 domains for polysaccharide chain
- Core polysaccharide – conserved across species,
attaches chain to phosphate on lipid a through
amine ester linkage - Species specific O-antigen, variable external
region
15
Q
Structure of the Gm (-) Cell Wall cont
A
- Lipid A region of LPS comprises outer layer of outer membrane
- Bacterial endotoxin, uses NAG instead of glycerol
- Responsible for symptoms associated with Gm (-) infections (fever, malaise, etc.) – more
on this later - Interior layer of outer membrane comprised of “typical” lipids
- Peptidoglycan layer beneath outer
membrane, much thinner than Gm (+)
layer, 1-2 sheets thick - Plasma membrane beneath
peptidoglycan, space between outer
and inner membrane referred to as
periplasm
16
Q
Why does the Gram Stain Work?
A
- Several theories, I’ll tell ya my favorite one
- Crystal violet is charged
- Outer membrane of Gm (-) has hydrophobic core
- Crystal violet never comes into contact with peptidoglycan
- Must in order for iodine mordant to exhibit affect
- Therefore, gm (-) bacteria are distained and must be counterstained
- Color following Gm staining allows you to infer the structure of the
cell wall
17
Q
Other Types of Microscopy
A
- Bright field microscopy useful but limited
- Obtain information regarding structure of cell wall and cell
morphology - Other microscopy methods allow gathering of different information
or increase the resolution - May used different forms of staining, optical mechanisms, or both to
increase contrast - Include phase contrast, dark field, fluorescent, differential
interference, atomic force, confocal, transmission electron, and
scanning electron microscopy
18
Q
Phase-contrast
A
- Two parts of the light beam are integrated
- One comes straight through the specimen
- The other is highly diffracted light collected from the edge of the
lens - Light is diffracted due to the fact that various intra- and extracellular structures have different refractive indexes
- Diffracted light subtracts from direct light giving the structures
with the greatest refractive index the darkest appearance
19
Q
Dark-Field Microscopy
A
- An opaque disk over middle of light source creates a donut-shaped
beam. - Only way for light to reach the specimen is to approach at an angle -
normally miss lens - Only light reflected by the specimen itself enters the lens
- Specimen appears light against a dark background
20
Q
Fluorescence
A
- A substance fluoresces when it
absorbs light at one wavelength,
and emits it at another - Can use fluorescent stains to
produce a bright object on dark
background - Stains can be general - i.e. DAPI,
stains DNA - Can be made highly specific by
conjugating fluorescent species to
antibodies
21
Q
Differential Interference Contrast Microscopy
A
- Requires polarizer to generate polarized light
- Polarized light passes through prism, split into multiple beams
- Individual beams pass through different structures of cell
- Different structures have different refractive index
- Beams taken out of phase
- Recombined when passing through objective lens
- Results in destructive interference, 3D image
22
Q
Atomic Force Microscopy
A
- Similar principle to STM
- Uses a metal carbon
composite probe - Probe traces surface of
specimen - Peaks and valleys recorded
- Generates 3D image
23
Q
Confocal
A
- Uses fluorescent stains for contrast
- A laser illuminates a very thin section of the specimen
- Laser allows use of pinhole aperture - less blurring
- Produces very clear images
- Can construct 3-D images by piling up optical sections
24
Q
Electron Microscopy
A
- Wavelengths of electrons smaller than photons
- This results in better resolution
- Uses electron beam instead of light
- Uses magnets to focus instead of lenses
25
TEM Protocol
* Specimen stained with osmium - very electron opaque
* Thin sections cut with diamond or sapphire blade
* Floated onto copper mesh, shadowed if necessary, stuck into scope
* Beam passes through thinly-sliced specimen
* Projects onto a phosphor screen that glows where
electrons hit
* Electron-opaque areas appear dark, areas that don’t
block electrons appear light
* Resolution allows up to 100,000 X magnification
26
SEM Protocol
* No sectioning, maybe some sputtering - a thin coating of tungsten or
gold may be sprayed over the specimen
* Beam strikes whole specimen
* Detector observes reflected electrons from an angle
* Provides 3D view of specimen, resolution allows up to 10,000X
magnification
27
Prokaryotic Cellular Morphology - Cocci
*Dipplococci - pairs of sphere shaped bacteria
* Streptococci - Chains of sphere shaped bacteria
* Tetrads - Planar clusters of 4 sphere shaped bacteria
* Sarcinae - Cube like clusters of 8 sphere shaped bacteria
* Staphylococci - Grape like clusters of sphere shaped bacteria
28
Prokaryotic Cellular Morphology - Bacilli
* Single bacillus - single rod shaped bacteria
* Diplobacilli - end on end pairs of rod shaped bacteria
- only seen shortly after cell division
* Streptobacilli - linear chains of rod shaped bacteria
* Cocobacilli - Very short rod like bacteria
29
Prokaryotic Cellular Morphology
Spiral Bacteria
* Vibrio - Bent rods
* Spirillum - helical shape with rigid bodies
* Spirochete - helical shape with flexible bodies
30
Uncommon Bacterial Shapes
* Star shaped bacteria - Stella
* Rectangular flat cells - Haloarcula
* Some species are triangular in shape
31
The Plasma Membrane
* Comprised of phospholipids
* Glycerol and phosphate groups hydrophilic
* Fatty acid groups hydrophobic
* Spontaneously assemble into bilayer
* Hydrophilic groups oriented toward aqueous environment
* Hydrophobic groups associate with each other to exclude water
* Thermodynamically favorable assembly
32
The Plasma Membrane cont
* Plasma membrane encompasses the cell
* Contain embedded proteins
* Can be peripherally associated or integral
* Provide structural integrity
* Facilitate transfer substances
* Not a static system - fluid mosaic model
Cholesterol
Diploptene
* Eukaryotic membranes contain cholesterol –
regulates fluidity, provides structural integrity
* Some prokaryotes use cholesterol like
molecules for same purpose - hopanoids
33
Archea Membranes
* Do not use fatty acids for hydrophobic interior, use isoprenes instead
* Unusual linkage between glycerol and isoprene – ether linkage
* Most life forms use ester linkage
* Some species have lipid bilayers, others have lipid monolayers
* Species with bilayers use phyntanyl
Archea Membranes
* Monolayers use biphytanyl or crenarcheol, twice as
long
34
Movement Across the Plasma Membrane
* Hydrophobic core of membrane limits diffusion, only small polar molecules
cross efficiently
* Macromolecules and ions dramatically retarded in transport, several
mechanisms assist, involve transmembrane transporters
Movement Across the Plasma Membrane
* Uniporter – transports one molecule across membrane along concentration gradient –
spontaneous
* Symporter – transports two distinct molecules
in the same direction, often uses proton motive
force
* Antiporter – transports two distinct molecules
in opposite directions, often uses proton motive
force
35
Group Translocation
* Glucose uptake as example – obtains energy from high energy phosphate bond in
phosphoenolpyruvate (PEP)
* Phosphate and energy transferred to Enz I, HPr, EnzIIa, Enz IIb, and Enz IIc sequentially
* Enz IIc – membrane spanning protein, binds glucose, transfers phosphate to glucose
* Energy transfer allows translocation into cell
36
The ABC Transporter System
* Requires three proteins – periplasmic carrier protein, transmembrane transprorter, and
cytoplasmic protein
* Cytoplasmic protein has ATP Binding Cassette (ABC) – capable of binding and hydrolyzing ATP
* Periplasmic protein binds macromolecule to be transported, interacts with periplasmic
domain of transmembrane transporting protein
* Causes conformational change in transporter,
transduced to ABC protein associated with
cytoplasmic domain
* Causes hydrolysis of ATP bound, energy
released used to transport macromolecule into
cell
37
Structures External to Cell Wall – Capsules and
Slime Layers
* Capsules - polysaccharide layer outside the cell
* Can be only loosely associated “slime layer”
* Important for virulence in many pathogens
* Involved in anti-phagocytic activity and/or attachment
* Essential for biofilm formation – structures formed during
growth under environmental conditions
38
Structures External to Cell Wall – Fimbriae, Pili, and Hami
* Fimbriae - short appendages used for attachment
* Consist of pilin
* Can be few at poles or many all over the cell
* Important for pathogenesis, allow colonization
* Pili - longer than fimbriae, consist of pilin
* May be used to join two cells together for exchange of genetic material
* Often referred to as sex pili
* Archea may use hamus (pleural hami) – short pili like structure terminating in hook like
structure
39
Inside the Cell – Carbon Storage Structures
* Excess carbon availability results in generation of inclusions – storage structures, visible with
specific types of microscopy
* Two common molecules used to store excess carbon – poly-b-hydroxybutyric acid (PHB) and
glycogen
Inside the Cell – Carbon Storage Structures
* PHB – lipid formed from b-hydroxybutyric acid, monomers
joined through ester linkages
* Glycogen – preferred energy storage unit
* Both can be used for energy, glycogen more efficient
* PHB also useful for carbon skeletons
40
Inside the Cell – Phosphate and Sulfur Storage Structures
* Excess phosphate leads to generation of granules, may be used for lipid and nucleotide
biosynthesis under starvation conditions
* Excess reduced sulfur (H2
S) oxidized to elemental sulfur So
* Accumulates in granules in periplasm
* Starvation conditions trigger oxidation of to Sulfate (SO4
2-
), used for biosynthesis of amino
acids
41
Gas Vesicles
* Used to regulate buoyancy, usually spindle shaped vesicles
* Vesicle not bordered by lipid membrane
* Two proteins prevent gas from escaping
* GvpA forms interlocking b-pleated sheets
* Forms boundary of vesicle
* GvpA “envelope” reinforced by GvpC
* Forms a-helices that run perpendicular to b-pleated sheets
42
Non-storage Inclusions – Magnetosomes
* Found in magnetotactic bacteria, inclusions containing iron
* Not a “true” storage structure – cannot be used for metabolism
* Allow bacteria to orient cell along magnetic poles
* Results in subsequent movement along poles, significance unknown
43
Endospores
* Dormant form of bacteria induced upon depletion of nutrients (nitrogen or carbon source)
* Highly resistant to harsh conditions (desiccation, heat, chemicals)
* Germinate if favorable conditions return
* Causes global change in gene expression - DIFFERENTIATION
* New enzymes, metabolites and structures produced
* Majority of vegetative structures vanish
* Requires alteration of transcriptional specificity of RNA polymerase
* Accomplished by use of alternate SIGMA FACTOR
44
Sporulation Process
* DNA replicates, moves to opposite poles of cell, septum forms at one end, generates FORE
SPORE
* Main cell mass engulfs fore spore generating SPORE MOTHER CELL - enclosed in two
membranes
* Peptidoglycan deposited between two
membranes, generates CORTEX
* Keratin like protein deposited outside
outer membrane generates spore COAT,
responsible for resistance to harsh
conditions
* EXOSPORIUM forms outside coat,
lipoprotein membrane containing
carbohydrates
45
Spore Germination
* Return of favorable conditions insufficient for germination
* Spore coat must be damaged (heat, abrasion, acidity, sulfhydryl containing compounds)
* Activates autolysin - degrades peptidoglycan in cortex
* Water taken up, enzymes degrade spore components
* Degradation of cortex generates spore protoplast
* Vegetative cell lacking mature peptidoglycan cell wall
* Undergoes biosynthesis of cell wall and other essential structures
* Requires all essential nutrients for growth
46
Structures External to Cell Wall – Flagella
* Long filamentous projections made of flagellin used to propel
bacteria
*4 types of arrangements
Monotrichous - single polar flagella
Amphitrichous - tufts of flagella at both poles
Lophotrichous – tufts of two or more flagella at one pole
Petritrichous - flagella distributed over entire
surface
47
Structures External to Cell Wall – Flagella cont 1
* Filament of flagella composed of flagellin
* Attached to cell wall by hook
* Hook attached to cell wall by L ring
* L ring embedded in outer membrane, not present in Gm (+)
bacteria
* L ring attached to P ring, embedded in peptidoglycan layer in
periplasm
* P ring attached to MS ring, associated with plasma membrane
* MS ring attached to C ring, exposed to cytosol
* Export apparatus attached to C ring, promotes assembly
48
Structures External to Cell Wall – Flagella cont 2
* MS/C rings surrounded by Mot proteins, creates strator
* Mot proteins comprise the motor, physically rotate MS/C rings
* Results in rotation of entire flagella
* Fli proteins sandwiched between MS and C rings, change
direction of flagella rotation
* Rotation in one direction initiates movement, switching
direction initiates tumbling, direction inducing movement vs.
tumbling ENTIRELY species dependent
* Uses proton motive force for rotation
49
Structures External to Cell Wall – Flagella cont 3
* Counterclockwise rotation results
in movement
* Clockwise results in tumbling
* Makes chemo- and photo-taxis
possible
* Attractants induce “running”
* Repellants induce “tumbling”
