1
Q
Fill in the blanks HOE
A
2
Q
Compound Light Microscopes
A
- Use visible light to illuminate cells
3
Q
Bright-field scope
A
- 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
4
Q
Fill in the blanks for a bright-field scope
A
5
Q
A
6
Q
Magnification
A
the ability to make an object larger
7
Q
Resolution
A
the ability to distinguish 2 adjacent objects as seperate and distinct (limit of resolution for light microscope is about 2 mewm/200 nm
8
Q
Microscope vs microscopy vs micrograph
A
microscope: object, microscopy: action, micrograph: resulting image
9
Q
Calculating magnification
A
ocular x objective
10
Q
How does resolution work?
A
- 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
11
Q
A
12
Q
As wavelength decreases, resolution..
A
improves
- because the shorter the wavelength the easier it is to fit through
13
Q
How to improve contrast in light microscopy
A
Staining!
14
Q
How does staining work?
A
- Using dyes that are organic compounds, they bind to specific cellular materials
- Some microbes already are pigmented (ex. chloropyll make a microbe green)
15
Q
Examples of common stains and their colours
A
Methylene blue - blue
Safranin - pink/red
Crystal violet - purple
16
Q
Chromophore
A
charged portion of a dye
17
Q
Simple staining
A
One dye used to colour specimen
18
Q
Basic dye vs acidic dye in simple staining
A
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
19
Q
A
20
Q
Differential stains
A
gram stain - separates bacteria into 2 groups based on cell wall structureg
21
Q
Gram positive vs gram negative in differential stains
A
gram positive: cells that retain a primary stain (purple)
gram negative: cells that lose the primary stain (take colour of counterstain - red/pink
22
Q
A
23
Q
Acid fast stain
A
- 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)
24
Q
Endospore stain
A
internal structures and super resistant to killing
- endospore - retains primary - green
- cells - counterstained - pink
- bacillus athracis
25
Disadvantages to stains
it kills the cells because its being dyed and heated - so we can't use it for motiltiy
26
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
27
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
28
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
29
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
30
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
31
Electron microscopy
- uses electrons instead of photons (light) to image cells and structures
- wavelength of electrons is much shorter than light - higher resolution
32
33
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
34
35
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
36
37
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
38
Coccus
plural: cocci
spherical
ex) streptococcus pyogenee
39
Bacillus
plural: bacilli
rod shaped
ex) e.coli
40
Spirillium
plural spirilla
spiral shaped
ex) spirillum volutans
41
1. cocci
2. bacillus
3. spirillum
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Spirochete
corkscrew
ex) treponema pallidum
43
Budding and appendaged bacteria
looks like a balloon
ex) caulobacter crescentus
- has the long part of attachment
44
Filamentous bacteria
ex) streptomyces griseus
45
1. spirochete
2. budding and appendaged bacteria
3. filamentous bacteria
46
What does morphology not predict
physiology, ecology, phylogeny
47
Selective forces in morphology
1. optimization for nutrient uptake
2. swimming motility in viscous environments or near surfaces
3. gliding motility
48
Prokaryotic sizes and examples
1. average - E.coli (1.0x3.0 mewm)
2. very small - mycoplasma genitalium (0.3 mewm)
3. very large - epulopiscium fishelsonii (80x600 mewm)
49
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
50
Cellular organisms less than ___ in diameter are unlikely
0.25 mewm
51
Open oceans tend to contain ___
small cells (0.2-0.4 mewm in diameter)
52
Why are pathogenic bacteria small
they are missing genes - get these functions of issing genes from hosts
53
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
54
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
55
56
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
57
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
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Integral vs peripheral membrane proteins
integral: firmly embedded in the membrane
peripheral: one portion anchored in the membrane
59
Archaeal Membranes
- ether linkages in phospholipids
- archael lipids lack fatty acids - isoprenes instead
60
61
Lipid monolayers and bilayers and heat
bilayer - not heat resistance
monolayer - heat resistance
62
What type of prokaryote are lipid monolayers found
hyperthermophilic archaea- likes high temps
63
Membrane functions
1. surrounds the cell - seperates cytoplasm from environment
2. highly selective permeable barrier - enables concentration of specific metabolites and excretion of wastes products (concentration gradient to represent the needs of the cell)
3. protein anchor - holds transport proteins in place
4. energy conservation - generation of proton motive force
64
Carrier-mediated transport systems
- shows saturation effect
- highly specific
65
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Uniporters, symporters, antiporters
1. transport in one direction across the membrane
2. cotransporters
3. transport a molecule across the membrane while simultaneously transporting another molecule in the other direction
67
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
68
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
69
ABC Transport systems
- uptake of organic compounds and inorganic nutrients and trace metals
- high substrate specificity - picky
70
Gram-negative ABC transport system
periplasmic-binding proteins and ATP-driven transport proteins
71
Gram-positive ABC transport system
substrate-binding lipoproteins (anchored to external surface of cell membrane) and ATP-driven transport proteins
72
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
73
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
74
Peptidoglycan
- species of bacteria seperated into 2 groups based on gram stain
- provides strength to cell wall
75
Do archaea have peptidoglycan
Naur!!!
76
Gram-positive vs gram-negative cell wall structure
Gram-negative: 2 layers - LPS and peptidoglycan
Gram-positive: 1 later - peptidoglycan
77
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
78
How can peptidoglycan vary
- 100 different structures
- vary in peptide cross-links or interbridge
79
Is an interbridge present in gram-negative bacteria
naur!
80
Gram-positive cell wall
90% peptidoglycan
- teichoic acids (acidic substances) embedded in cell wall
81
Lipoteichoic acids
teichoic acids covalently bound to membrane lipids
82
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
83
prokaryotes that lack cell walls and what replaces their cell walls?
- Mycoplasmas - sterols in cytoplasmic membrane
- Thermoplasma - lipoglycans in membrane
84
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
85
periplasm
- space located between cytoplasmic and outer membrane
- gel-like consistency
- has many proteins
86
porins
channels for movement of hydrophilic low-molecular-weight substances
87
Bacteria outer membrane summary
88
Cell wall and gram stain relationship
1. Stained with crystal violet - it gets stuck inside cell
2. Flushed with alcohol - + not extracted, - is extracted
3. 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
4. stained with safranin - + purple is darker than pink so u cant see it, - is stained pink and red
89
Archael cell walls
- no peptidogly
- no outer membrane
- instead they have pseudomurein
- some archaea dont have pseudomurein
90
What is pseudomurein composed of
- polyscharride
- N-acetylglucosamine
- N-acetyltalosaminuronic acid
- beta 1,3 linkage
91
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
92
S-layer
- archaea cell wall type
archaea and bacteria
- proteins and glycoprotein
- paracrystalline structure (hexagonal, tetragonal, trimer)
- some archaea only have S-layer
93
Why are archaea resistant to lysozyme and penicilin?
They lack peptidoglycan
94
Cytoplasm
material bounded by plasma membrane
95
Protoplast
PM and everything within
- macromolecules
- soluble proteins
- DNA and RNA
96
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)
97
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
98
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
99
Capsules and slime layers
- expresses sometimes
- polysaccharides and protein layers
- think or thick
- attachment to surfaces
- protects against phagocytosis
- resist drying out
100
biofilm
- community of bacteria associated with a surface
- stronger together
101
fimbriae
- filamentous protein structures
- stick to surfaces or form pellicles
102
pili
- filamentous protein structure
- longer than fimbriae
- surface attachment
- gene exchange between cells (for better genes) - horizontal
- type 4 pili for twitching motility
103
cell inclusion bodies
visible aggregates in cytoplasm
104
carbon storage polymers
- poly-B-hydroxybutyric acid - lipid storage
- glycogen - glucose polymer
105
inorganic inclusions
- polyphosphate granule - volutin - storage of phosphate and energy
- sulfur globules - storage of sulfur used in energy generation
106
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
107
gas vesicles
- give buoyancy
- spindle-shaped, gas-filled structures
- made of proteins
- function by decreasing cell density
- impermeable to water
108
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
109
how to kill endospore
autoclave - pressure and heat
110
what kind of gram bacteria produces endospores
gram positive
111
endospore growth
- vegetative - capable of normal growth (metabolically active)
112
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
113
What can endospores resist
1. boiling for hours
2. uv radiation
3. chemical disinfectants
3. drying
4. age
114
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
115
flagella
- hollow protein filaments
- for motility
- must be stained to view
116
monotrichous
single flagella
1. polar - on the side
2. sub polar on bottom or top
117
amphitrichous
flagella at opposite ends
118
lophotrichous
multiple flagella in a single tuft
119
peritrichous
flagella distributed around the cell
120
flagella structure
1. filament
2. hook
3. basal body
121
filament in flagella
- helical protein 20 mewm long
- composed of identical protein subunits - flagellin
122
hook in flagella
- flexible coupling between filament and basal body
123
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
124
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
125
126
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
127
Peritrichous vs polar cells swimming motions
peri - move slowly in a straight line
polar - more more rapidly and spin more
128
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)
129
Taxis
directed movement of cells in response to chemical or physical gradients
130
chemotaxis, phototaxis, aerotacis, osmotaxis, hydrotaxis
chemo - response to chemicals
photo - light
aero - oxygen
osmo - ionic strength
hydro - water
131
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
132
Measuring chemotaxis
- Measured by inserting a capillary tube containing an attractant or a repellants in a medium of motile bacteria
- seen under a microscope
133
Eukaryote cell size
- lower surface area to volume ratio
- larger than pro
134
Key differences in pro and euk
135
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
136
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
137
- 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
138
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)
138
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
139
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
140
icosahedral capsids
-regular geometric shape with 20 triangular faces
- symmetry
- protomers aggregate to form capsomeres
hpv
141
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
142
Nucleocytoplasmic large DNA viruses
- viruses with complex multi-layered structure
- larger than some bacteria
-mimivirus
143
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)
144
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
145
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
146
Viral replication cycle
1. Absorption - attachment to host cell - involves specific receptors on the host cell surface
2. 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
3. Synthesis of viral nucleic acids and protein - viral genes are expressed and viral proteins are synthesized - viral genome is replicated
4. Assembly of new virions - viral proteins are assembled into capsids and then genomes are packaaged into nucleocapsids - viruses don't reproduce by division
5. 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
147
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
Microbiology
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