2. Cell Structure And Microscopy Flashcards
Fill in the blanks HOE
Compound Light Microscopes
- Use visible light to illuminate cells
Bright-field scope
- Type of light microscope
- Visualized by the differences in contrast between specimen and surroundings
- Two sets of lenses from the image: objective lens (10x-100x) and ocular lens (10x-20x)
- Max: 2000x
Fill in the blanks for a bright-field scope
Magnification
the ability to make an object larger
Resolution
the ability to distinguish 2 adjacent objects as seperate and distinct (limit of resolution for light microscope is about 2 mewm/200 nm
Microscope vs microscopy vs micrograph
microscope: object, microscopy: action, micrograph: resulting image
Calculating magnification
ocular x objective
How does resolution work?
- Two points can be distinguished if they are atleast .2 mewm apart
- light must pass between two points for them to be viewed as seperate objects
As wavelength decreases, resolution..
improves
- because the shorter the wavelength the easier it is to fit through
How to improve contrast in light microscopy
Staining!
How does staining work?
- Using dyes that are organic compounds, they bind to specific cellular materials
- Some microbes already are pigmented (ex. chloropyll make a microbe green)
Examples of common stains and their colours
Methylene blue - blue
Safranin - pink/red
Crystal violet - purple
Chromophore
charged portion of a dye
Simple staining
One dye used to colour specimen
Basic dye vs acidic dye in simple staining
Basic - positively charged chromophore - binds to negatively charged molecules on cell surface
Acidic - negatively charged chromophore - repelled by cell surface - stains background - good for looking at cell shape and size
Differential stains
gram stain - separates bacteria into 2 groups based on cell wall structureg
Gram positive vs gram negative in differential stains
gram positive: cells that retain a primary stain (purple)
gram negative: cells that lose the primary stain (take colour of counterstain - red/pink
Acid fast stain
- for acid fast bacteria
- detects mycolic acid in the cell wall of mycobacterium
- mycobacterium - retains a primary stain (pink)
- everything else - colour of counterstain (blue)
Endospore stain
internal structures and super resistant to killing
- endospore - retains primary - green
- cells - counterstained - pink
- bacillus athracis
Disadvantages to stains
it kills the cells because its being dyed and heated - so we can’t use it for motiltiy
Phase-contrast microscopy
- phase ring amplifies differences in the refractive index of cell and surroundings - doesn’t change the bacteria
- used for live samples
- dark cells with a light background
Dark field microscopy
- less common
- specimen is illuminated with a hollow cone of light
- only refracted light enters the objective
- specimen appears as a bright object on a dark background
- used to observe bacteria that doesn’t stain well
- triponema pallium
Fluorescence microscopy
- used to visualize specimens that fluoresce
- emit light of one color when illuminated with another colour of light
- cells may fluoresce naturally (absorbs light at 430 nm - blue-violet and emits at 670 bm - red)
- cells may fluoresce after staining with dye
cyanobacteria
Differential Interference contrast (DIC) microscopy
- uses a polarizer to create 2 distinct beams of polarized light
- gives structures a 3D appearance (endospores, vacuoles, granules)
- Structures not visible by bright-field microscopy are sometimes visible with this
Confocal scanning laser microscopy
- uses a computerized microscope coupled with a laser source to generate 3D image
- computer can focus the laser on single layers of the specimen
- different layers can be compiled for a 3D image
Electron microscopy
- uses electrons instead of photons (light) to image cells and structures
- wavelength of electrons is much shorter than light - higher resolution
TEM
transmission electron microscope
- electron beamed focused on specimen by a condenser - magnets used as lenses
- electrons that pass through the specimens are focused by two sets of lenses - compound microscope
- electrons strike a fluorescent viewing screen
- goes through cell
Tem magnification, resolution, size, staining
- high magnification and resolution (0.2 nm)
- specimen is thin (20-60 bm)
- must be stained with metals (lead or uranium), bind to cell structure to make it more electron dense, to see visualization of structures at molecule level
SEM
- specimen is coated with a thin film of heavy metal (gold)
- an electron beam scans the object
- scattered electrons are collected by a detector and an image is produced
- allows an accurate 3D image of the specimens surface
- looks at surface of cell
Coccus
plural: cocci
spherical
ex) streptococcus pyogenee
Bacillus
plural: bacilli
rod shaped
ex) e.coli
Spirillium
plural spirilla
spiral shaped
ex) spirillum volutans
- cocci
- bacillus
- spirillum
Spirochete
corkscrew
ex) treponema pallidum
Budding and appendaged bacteria
looks like a balloon
ex) caulobacter crescentus
- has the long part of attachment
Filamentous bacteria
ex) streptomyces griseus
- spirochete
- budding and appendaged bacteria
- filamentous bacteria
What does morphology not predict
physiology, ecology, phylogeny
Selective forces in morphology
- optimization for nutrient uptake
- swimming motility in viscous environments or near surfaces
- gliding motility
Prokaryotic sizes and examples
- average - E.coli (1.0x3.0 mewm)
- very small - mycoplasma genitalium (0.3 mewm)
- very large - epulopiscium fishelsonii (80x600 mewm)
Advantages to being small
small cells have more surface area relative to cell volume than large cells (higher surface to volume ratio)
- support greater nutrient exchange per unit cell volume
- tend to grow and adapt faster than larger cells
Cellular organisms less than ___ in diameter are unlikely
0.25 mewm
Open oceans tend to contain ___
small cells (0.2-0.4 mewm in diameter)
Why are pathogenic bacteria small
they are missing genes - get these functions of issing genes from hosts
Membrane is made out of
- phospholipid bilayer
- hydrophobic: fatty acids point inward
- hydrophilic: glycerol-phosphate and points to external environment
- can exist in many different chemical forms as a result of variation in the groups attached to the glycerol backbone
Phospholipid structure
ester phospholipids:
- glycerol
- 2 fatty acids
- phosphate
- optional side chain
amiphipathic: has polar and non-polar characteristics
polar: molecule carries full or partial charge - hydrophilic
non-polar: molecule is uncharged - hydrophobic
How are these stabilized
- 8-10nm wide
- embedded proteins
- stabilized by hydrogen bonds and hydrophobic interactions
- Mg and Ca ions help stabilize the membrane through ionic bonds with negative charges on the phospholipids
Membrane proteins in gram negative
in gram-negative bacteria;
- interacts with proteins that bind substrates or process large molecules for transport
- interacts with proteins involved in important cell functions like energy-yielding reactions
Integral vs peripheral membrane proteins
integral: firmly embedded in the membrane
peripheral: one portion anchored in the membrane
Archaeal Membranes
- ether linkages in phospholipids
- archael lipids lack fatty acids - isoprenes instead
Lipid monolayers and bilayers and heat
bilayer - not heat resistance
monolayer - heat resistance
What type of prokaryote are lipid monolayers found
hyperthermophilic archaea- likes high temps
Membrane functions
- surrounds the cell - seperates cytoplasm from environment
- highly selective permeable barrier - enables concentration of specific metabolites and excretion of wastes products (concentration gradient to represent the needs of the cell)
- protein anchor - holds transport proteins in place
- energy conservation - generation of proton motive force
Carrier-mediated transport systems
- shows saturation effect
- highly specific
Uniporters, symporters, antiporters
- transport in one direction across the membrane
- cotransporters
- transport a molecule across the membrane while simultaneously transporting another molecule in the other direction
Simple transport example
Lac permease of E.Coli
- lactose is transported into e coli by lac permease a symporter that moves 2 molecules (lactose and H+) across a membrane in the same direction
- energy-driven
Group translocation example
phosphotransferase system in E. Coli
- sugar is phosphorylated during transport across the membrane
- moved glucose, fructose and mannose
- PEP donates a P to a phosphorelay system
- P is transferred through a series of carrier proteins and deposited onto the sugar as its brought into the cell
ABC Transport systems
- uptake of organic compounds and inorganic nutrients and trace metals
- high substrate specificity - picky
Gram-negative ABC transport system
periplasmic-binding proteins and ATP-driven transport proteins
Gram-positive ABC transport system
substrate-binding lipoproteins (anchored to external surface of cell membrane) and ATP-driven transport proteins
Cell wall in bacteria and archaea
- outside the cell membrane
- rigid
- determines cell shape
- not a major permeability barrier
- porous to most small molecules
- protects the cell from osmotic changes
Functions of the cell wall
- prevents cell expansion (protects from osmotic lysis)
- protects against toxic substances (large hydrophobic molecules)
- pathogenicity - helps evade host immune system and bacterium stick to surfaces
Peptidoglycan
- species of bacteria seperated into 2 groups based on gram stain
- provides strength to cell wall
Do archaea have peptidoglycan
Naur!!!
Gram-positive vs gram-negative cell wall structure
Gram-negative: 2 layers - LPS and peptidoglycan
Gram-positive: 1 later - peptidoglycan
What is peptidoglycan made of
- A polysaccharide
- N-acetylglucosamine
- N-acetylmuramic acid
- amino acids
- lysine or diaminopimelic acid (DAP)
- cross-linked differently in gram+ and gram-
- forms glycan tetrapeptide
- beta 1,4 linkage
How can peptidoglycan vary
- 100 different structures
- vary in peptide cross-links or interbridge
Is an interbridge present in gram-negative bacteria
naur!
Gram-positive cell wall
90% peptidoglycan
- teichoic acids (acidic substances) embedded in cell wall
Lipoteichoic acids
teichoic acids covalently bound to membrane lipids
Peptidoglycan
- backbone formed of ___ and ___ connected by _____________
- crosslinks formed by _________
- peptidoglycan strand is ________ and allows for ______________ (It needs this to ____________)
- some cell walls can be _______ layers thick
- backbone formed of NAM and NAG connected by glycosidic bonds
- crosslinks formed by peptides
- peptidoglycan strand is helical and allows for 3d crosslinking. it needs this to hold them together
- some cell walls can be 50-100 layers thick
prokaryotes that lack cell walls and what replaces their cell walls?
- Mycoplasmas - sterols in cytoplasmic membrane
- Thermoplasma - lipoglycans in membrane
lipopolysaccharide layer
- a lipid with many sugars
- The outer membrane
- Total cell wall contains 10% peptidoglycan
- consists of core polysaccharide and O-polysaccharide
- LPS replaces most of phospholipids in outer half of outer membrane
- Lipid A (endotoxin) is toxic
periplasm
- space located between cytoplasmic and outer membrane
- gel-like consistency
- has many proteins
porins
channels for movement of hydrophilic low-molecular-weight substances
Bacteria outer membrane summary
Cell wall and gram stain relationship
- Stained with crystal violet - it gets stuck inside cell
- Flushed with alcohol - + not extracted, - is extracted
- gram positive bacteria have thick cell walls - it becomes dehydrated during the alcohol step so pores in wall close and prevents colour from escaping
gram negative bacteria - alcohol penetrates OM and colour is extracted from cell so its clear - stained with safranin - + purple is darker than pink so u cant see it, - is stained pink and red
Archael cell walls
- no peptidogly
- no outer membrane
- instead they have pseudomurein
- some archaea dont have pseudomurein
What is pseudomurein composed of
- polyscharride
- N-acetylglucosamine
- N-acetyltalosaminuronic acid
- beta 1,3 linkage
Differences in peptidoglycan and pseudomurein
Peptidoglycan:
- N-acetylmuramic acid
- beta 1,4 linkage
- structure has L and D
Pseudomurein:
- N-acetyltalosaminuronic acid
- beta 1,3 linkage
- structure it has L only
S-layer
- archaea cell wall type
archaea and bacteria - proteins and glycoprotein
- paracrystalline structure (hexagonal, tetragonal, trimer)
- some archaea only have S-layer
Why are archaea resistant to lysozyme and penicilin?
They lack peptidoglycan
Cytoplasm
material bounded by plasma membrane
Protoplast
PM and everything within
- macromolecules
- soluble proteins
- DNA and RNA
Protein functions
- enzymes that catalyze chemical reactions
- transport proteins - move other molecules across membranes
- structural proteins - help determine shape of cell and cell division
- proteins are made of polypeptides (a long polymer of amino acids joined by peptide bonds)
Nucleoid
- region that containes genome
- typical bacterial genome: single circular double stranded DNA chromosome
- may have one or more plasmids - smaller circular dsDNA, self-replicating, carry non-essential genes (selective advantage)
- DNA: carries genetic info of all cellsR
Ribosome
- site of protein synthesis
- bacteria have 70s ribosome
- 30s subunit (small subunit) - protein and 16s rRNA
- 50s subunit (large subunit) - protein and 23s and 5s rRNA
- cytoplasmic ribosome that make cytoplasmic proteins
- PM associated with ribosomes - membrane proteins that are exported from the cellc
Capsules and slime layers
- expresses sometimes
- polysaccharides and protein layers
- think or thick
- attachment to surfaces
- protects against phagocytosis
- resist drying out
biofilm
- community of bacteria associated with a surface
- stronger together
fimbriae
- filamentous protein structures
- stick to surfaces or form pellicles
pili
- filamentous protein structure
- longer than fimbriae
- surface attachment
- gene exchange between cells (for better genes) - horizontal
- type 4 pili for twitching motility
cell inclusion bodies
visible aggregates in cytoplasm
carbon storage polymers
- poly-B-hydroxybutyric acid - lipid storage
- glycogen - glucose polymer
inorganic inclusions
- polyphosphate granule - volutin - storage of phosphate and energy
- sulfur globules - storage of sulfur used in energy generation
magnetosomes
- magnetic inclusions
- intracellular granules that give the cell magnetic properties
- allows it to orient itself in a magnetic field
- bacteria migrate along earths magnetic magnetotaxis
gas vesicles
- give buoyancy
- spindle-shaped, gas-filled structures
- made of proteins
- function by decreasing cell density
- impermeable to water
endospores
- highly differentiated cells resistant to heat, harsh chemicals and radiation
- hibernating stage of bacterial life cycle
- ideal for dispersal via wind, water or animal gut
how to kill endospore
autoclave - pressure and heat
what kind of gram bacteria produces endospores
gram positive
endospore growth
- vegetative - capable of normal growth (metabolically active)
protective features of endospores
- Layers
1. spore coat and cortex for protection against chemicals, enzymes, physical damage and heat
2. two membranes - pearmeability barriers against chemicals - Core
3. dehydrated - protects against heat
4. ca-dipicolinic acid and SASPs (small acid soluble proteins) that protect against DNA damage
What can endospores resist
- boiling for hours
- uv radiation
- chemical disinfectants
- drying
- age
Stage 1. Asymmetric cell division
- DNA replicates
- identical chromosomes pulled to opposite ends of the cell
Stage 2. Septation
- divides cell into 2 unequal compartments - both have their own chromy
- forespore
- mother cell
Stage 3. Mother engulfs forespore
- forespore surrounded by 2 membrances
Stage 4. formation of the cortex
- thick layers of peptidoglycan form between 2 membranes
- highly cross-linked layer (core)
- loosely cross-linked layer (cortex)a
Stage 5. coat synthesis
- protein layers surround the core wall
- spore coat
- exosporium (found in some G+ and non-essential)
- protect the spore from chemicals and enzymes
- accumulates in spore; calcium, dipicolinic acid and small acid soluble proteins (SASPs) accumulate in the core to help stabilize DNA
Stage 6. endospore matures
- core is dehycrated
- 10-30% of a vegetative cells water content
Stage 7. mother cell is lysed
- mother cell disintegrates
- mature spore is released
flagella
- hollow protein filaments
- for motility
- must be stained to view
monotrichous
single flagella
1. polar - on the side
2. sub polar on bottom or top
amphitrichous
flagella at opposite ends
lophotrichous
multiple flagella in a single tuft
peritrichous
flagella distributed around the cell
flagella structure
- filament
- hook
- basal body
filament in flagella
- helical protein 20 mewm long
- composed of identical protein subunits - flagellin
hook in flagella
- flexible coupling between filament and basal body
basal body in flagella
consist of central rod that passes through a series of rings
1. L - LPS layer
2. P - peptidoglycan
3. MS - membrane
4. C - cytoplasm - associated with membrane
Energy in flagella
-energy to turn the flagella comes from the proton motive force (PMF)
- gradient of protons (H+) across the cytoplasmic membrane - high H+ on outside and low H+ on inside
- MOT proteins form a channel that allows H+ to move into cytoplasm
- flagellum turns like a propellar to drive cell forward
flagellar synthesis
- several genes are required for flagellar synthesis and motility
- MS ring is made first
- other proteins and hooks are made next
- filament grows from tip
Peritrichous vs polar cells swimming motions
peri - move slowly in a straight line
polar - more more rapidly and spin more
Gliding motility
- flagella independent motlity
- slower and smoother than swimming
- needs surface contact
- mechanisms: type 4 pili for twitching (ATP hydrolysis) and gliding specific proteins (proton motive force)
Taxis
directed movement of cells in response to chemical or physical gradients
chemotaxis, phototaxis, aerotacis, osmotaxis, hydrotaxis
chemo - response to chemicals
photo - light
aero - oxygen
osmo - ionic strength
hydro - water
Example of chemotaxis
- E.coli
- bacteria repsonded to temporal not spatial difference in chemical concentration
- run and tumble behavior
- attractants and repellants are sensed by chemoreceptors
- the net movement - run more and tumble less to attractant
Measuring chemotaxis
- Measured by inserting a capillary tube containing an attractant or a repellants in a medium of motile bacteria
- seen under a microscope
Eukaryote cell size
- lower surface area to volume ratio
- larger than pro
Key differences in pro and euk
Nucleus, chloroplast and mitochondria
nucleus - genetic info - multiple linear dsDNA chromosomes
chloroplasts - site of photosynthesis, chlorophyll, surrounded by 2 membranes, DNA and ribosomes (70s)
mitochondria - site of respiration and oxidative phosphorylation, surrounded by 2 membranes. DNA and ribosomes (70s)end
Endosymbiotic Hypothesis
mitochondria and chloroplasts evolved from bacteria
1. semi-autonomous - make their own DNA
2. circular choromsomes that lack histones
3. 70S ribosomes
4. 2 membranes
5. outer membrane has porins
- mitochondria and chloroplast mostly related to and why
mitochondria - most closely related to Rickettsia, proteobacteria, obligate intracellular pathogens
chloroplasts - most closely related to cyanobacteria, blue green algae
Comparison of 16s rRNA gene sequences
Viral genome, shape and size, strand number,
- DNA or RNA - never both
- single stranded or double stranded
- circular or linear
- can be in several pieces - segmented
- genome size:
small - 3.6 kb for ssRNA viruses (3 genes)
largest 150kbp for some dsDNA viruses (>100 genes)
Viruses
- non-living so technically not a microorganism
- acellular infectious particles
- obligate intracellular pathogens
- reproduce only inside living cell
- lack independent metabolism
- composed of at least 2 parts - nucleic acid genome (DNA or RNA) and protein code (capside) - nucleocapside
- some viruses have an envelope
- gets the rest of their parts from their host cell
capsid
protein coat that surrounds the genome
- allows transfer of viral genome between host cells
- made of identical polypeptides - protomer
- helical capsids - protomers form a spiral cylinder, nucleic acid genome coiled inside
ex) tobacco
icosahedral capsids
-regular geometric shape with 20 triangular faces
- symmetry
- protomers aggregate to form capsomeres
hpv
binary capsids
- geometric head with ana attached helical tail
- genome is carried in a polyhedral head, helical tail is used to inject DNA into a host cell
-t4 in ecoli
Nucleocytoplasmic large DNA viruses
- viruses with complex multi-layered structure
- larger than some bacteria
-mimivirus
envelope
a lipid bilayer surrounding the nucleocapsid that was acquired from the host membrance
- consists of host lipids and viral proteins - spikes
- flexible helical capsid, surrounded by an envelope
- 2 major spikes: hemaglutanin (H) and neuraminidase (N)
viruses - host range
- attach to specific receptors
- viruses infect all domains of life
- bacteriophage (phage) - viruses that infect bacteria
- animal viruses - infect and multiply only inside of animal cells - causes benign tumors
viruses - host range - how many attachments?
- most viruses are specific to a single host species
- virus must attach to specific receptors on the host cell surface - ex) HIV binds to CD4
chemoreceptor on surface of some human immune system cells - some viruses infect more than 1 specie ex) influenza
Viral replication cycle
- Absorption - attachment to host cell - involves specific receptors on the host cell surface
- Penetration and uncoating - entry into a host cell - bacteriophage (usually inject their nucleid acid into the cell) - leave the capside outside the cell as a ghost
- Synthesis of viral nucleic acids and protein - viral genes are expressed and viral proteins are synthesized - viral genome is replicated
- Assembly of new virions - viral proteins are assembled into capsids and then genomes are packaaged into nucleocapsids - viruses don’t reproduce by division
- Release of new virions by 1. naked viruses accumulare eventually lysing the host cell to release progeny - lytic infection or 2. enveloped viruses are usually released by budding - virions push through the cytoplasmic membrane without killing the host cell - persistent infection
Entry by animal viruses
- fusion with plasma membrane (envelope viruses only)
- endocytosis - bidning to specific receptors triggers normal endocytic activity
- once inside - the capside is removed - viral genome is released into the cell
neuraminidase allows new virions to exit the host cell
- hemagglutanin allows viruses to absorb to the next host
