Lecture #2 - Cell Structure & Function Flashcards
What are the relative sizes of objects & what microscope? Review slide 2
done
Large protozoa
euk microbe
- has euk cell structure
- larger than a prok, but smaller than a plant
- light microscope
RBC’s
human cell
- have to go 1 by 1 (small)
- @ maturity, they lose all their internal compartments to be smaller
- no organelles
Describe chloroplast
used to be prok. syn. bacteria (unicellular bacterium) which is why its large
- according to endosymbiotic theory
Mitochondria
used to be prok. syn. bacteria (unicellular bacterium) which is why it’s large
- according to endosymbiotic theory
- an organelle, inside of Euk cell
Describe Chlamydia
bacteria
- OBLIGATELY INTRACELLULAR (sounds like a virus)
- smaller than bacteria (b/c cell provides a lot for it)
- go into Euk cell to facilitate infection (goes into RT, & cytoplasm of cell where it’ll grow)
- antiobiotic that goes into ECF & cytoplasm of cell has GOOD TISSUE PENETRATION (blood –> tissue –> ECF –> cytoplasm to find target)
- DON’T KNOW HOW TO GROW IT (need a cell, so have to use nucleic acid testing (looking for genetic material)
- had ribosomes (& lots of cellular material) that made it large but lost a lot to make it smaller
If someone had chlamydia & got a swab. After its been spread on petri dish & incubated, will there be chlamydia growth the next day?
NO - b/c that growth med. will not be what they need
- still don’t know how to grow it
Describe rickettsia
an INTRACELLULAR ORGANISM
- ancestor of mitochondria
- goes into a cell
- may have got trapped to act like a mitochondria (replicated)
Describe viruses
OBLIGATE INTRACELLULAR parasites
- small packet of genetic material & bare min. needed for life cycle
Describe Ribosomes
made up of protein & rRNA
- organelle that doesn’t originate from cell - smaller
Order from largest to smallest (relative sizes of objects)
- Dog
- Human heart
- Tick
- Human egg
- Large protozoa
- RBC
- Chloroplast
- Bacteria (prok. uni - but exceptions)
- Mitochondrion
- Rickettsia
- Chlamydia
- Virsuses
- Ribosomes
- Proteins
- Diameter of DNA
- AA’s
- Atoms
What can be seen with the unaided human eye?
tick, human heart, dog
What can be seen with the compound light microscope?
Chlamydia, rickettsia, mitochondrion, bacteria, chloroplast, RBC, large protozoa, human egg, tick
What can be seen with the scanning electron microscope?
Ribosomes, viruses, chlamydia, rickettsia, mitochondrion, bacteria, chloroplast, RBC, Large protozoa, human egg, tick
What can be seen with a transmission electron microscope?
AA’s, diameter of DNA, proteins, ribosomes, viruses, chlamydia, rickettsia, mitochondrion, bacteria, chloroplast, RBC, large protozoa, human egg
What does a compound light microscope use?
visible light to illuminate cells
- light bulb
Light bulb
= low energy source to illuminate specimen, therefore limitations
What are the many different types of light microscopy?
- Bright-field
- Phase-contrast
- Dark-field
- Fluorescence
What is a Bright-field scope light microscope?
• Specimens are visualized because of differences in contrast between specimen (cell of interest) and surroundings (background they’re on)
- DARK cells on BRIGHT background
Why do we call the bright-field scope a compound light microscope?
b/c it compounds the magn. that the 1st lens is giving you by further magnifying it through a 2nd lens
- Two sets of lenses form the image
- Objective lens (can choose/change) (usually 10x -100x mag.) & ocular lens (can’t change) (usually 10x – 20x mag.)
- Total magnification = objective magnification ✕ ocular magnification
- Maximum magnification is ~2,000✕
Condenser
creates a beam of light so it’s condensed/focused to be able to move through the microscope slide
Describe the magnification light path
- Light from light source
- Condenser - focuses it into a beam so it’s interacting with the specimen
- Once the specimen comes through the objective lens (10X, 40X, or 100X (oil)), it’s inverted in position & magnified to whatever you chose
- When the specimen comes through the ocular lens (10X) it is inverted again to OG position & magnified further, so even larger
- Then you see it at either 100X, 400X, 1000X
Magnification
the ability to make an object larger
- e- microscopes are 2000x magnification or more
Resolution
(AKA resolving power)
the ability to distinguish two adjacent objects as separate and distinct
- the amount of light passing b/t will create a clear image & how much space is needed to do that is the resolving power
think: 2 hands touching & able to see 2 hands (not perfectly lined up)
What is the limit of resolution for light microscope?
about 0.2 μm
- MIN distance that 2 objects need to be apart from 1 another, in order for that microscope to show you separate objects
- if those 2 objects are less than that distance apart (closer together), the microscope cannot get energy source that it’s using b/t them, so you see a blurry image (not separate objects)
Limit of resolution for light microscope is about 0.2 μm, what does this mean? What happens if it is smaller or bigger?
If we report a resolving power/limited resolution = to 0.2 μm. That means, this microscope with the energy source it’s using can provide light that can only get through spaces that are AT LEAST 0.2 μm
- anything BIGGER works too
- anything smaller, the light CAN’T fit, therefore can’t energize sample or provide a clear image
If we had a better microscope, what would we expect the limit of resolution value to be?
SMALLER - b/c then those 2 objects can be even closer together & you can still see a clear image (ex: e- microscopes)
How do we calculate magnification?
- Magnification = ocular x objective
- ex. Ocular = 10x, objective = 40x
- Magnification = 10 40 = 400x
Resolution explained
- The ability of a lens to distinguish small objects that are close together
- Ex) resolving power of 0.2μm
- Two points can be distinguished if they are at least 0.2 μm apart
- Light must pass between two points for them to be viewed as separate objects (providing clarity)
- As wavelength decreases resolution improves
Wavelength & energy are _______
INVERSELY PROPORTIONAL
Shorter wavelength =
↑ ENERGY
↑ RESOLUTION
Longer wavelength
↓ ENERGY
↓ RESOLUTION
Ex’s of ↑ energy - shorter wavelengths
- gamma rays
- x-rays
- ultra-violet rays
↑ energy - shorter wavelengths =
DANGEROUS b/c energy it carries can be transferred all the way to DNA for ex & can result in damage/mutation that can lead to cancer
- ↑ energy radiation is dangerous b/c energy it carries will then be transferred to other objects its in contact with
Ex’s of ↓ energy - longer wavelengths
- infrared rays
- microwaves
- radio waves
↓ energy - longer wavelengths =
safer
If you have 2 microscopes, both with the same magnification, but diff. resolving powers. Will they provide the same image?
NO - b/c as you magnify, it doesn’t mean the resolution maintains clarity
- ex: zooming on phone doesn’t mean it will still be clear
GREATER magnification…
DOESN’T mean/guarantee resolution will also increase; dependent on microscope you chose
Throw ink-covered objects at target (“E”):
- Basketballs - longest wavelength
- Tennis balls - slightly shorter wavelength
- Jelly beans
- Beads - shortest wavelength
- CANNOT fit b/t arms, poor resolution
- Fit b/t arms, resolution improves
3 & 4. As DIAMETER of objects thrown DECREASES, GREATER NUMBERS pass b/t the arms & the RESOLUTION INCREASES (no size limitation - ton of clarity)
Improving contrast results in…
a better final image
- STICK OUT BETTER, BETTER CLARITY & you can see better dets of cell
Staining improves _____
CONTRAST
How does staining improve contrast?
• Dyes are organic compounds (carbon containing CH2-COO-) that bind to specific cellular materials (inside the cell)
Ex’s of common stains:
methylene blue, safranin, and crystal violet
What are the 2 types of staining?
- Simple staining
2. Differential stains
Simple staining
One dye used to color specimen
- just shows if something is THERE or NOT - 1 size fits all (1 colour only)
think: taking attendance at exam, are you there or not?
Chromophore
colored portion of a dye
- colour your cell will appear as a conseq. of that dye adhering to portions of the cell or cellular structures
Ex: red chromophore
What are the 2 types of simple stains/dyes?
- Basic dye
2. Acidic dye
A living cell, whether it’s a bacterial cell, fungal cell or human cell, will….
ALWAYS HAVE A NET (-) CHARGE
- if you apply a basic stain it will adhere
- if you apply an acidic stain it will repel
Basic dye
positively charged chromophore
• Binds to negatively charged molecules on cell surface
- (+) at pH=7
Acidic dye
negatively charged chromophore
• Repelled by cell surface
• Used to stain background (cells will stick out)
• Negative stain
- (-) at pH=7
Ex of basic stain:
crystal violet - cells are violet
Ex of acidic stain:
nigrosin - background is purple ish
How to prepare samples for staining
- PREPARING A SMEAR
- Spread culture in THIN film over slide - a. Dry in air
OR
b. HEAT FIXING & STAINING
- Pass slide through flame to heat fix
(think: cleaning pan next day - hard to get off, dehydrating sample so it is stuck on slide, therefore it doesn’t get rinsed off in next step)
- Flood slide with stain (engaging with cells or background - depending on if basic/acidic) - Microscopy
- Place drop of oil on slide; examine with 100X objective lens
- not gonna get differentiation, just able to see if it’s there
Gram - & gram + will BOTH…
be (-)ly charged on their external surface
What is the difference b/t gram + & gram -?
architecture of their cell wall is different
The Gram Stain
a differential stain; a staining procedure where you can create differences based on the cell structure, that allow you to identify if the bacterium that you have in your sample is gram + or gram -
What are 2 reasons to do the gram stain?
- Narrows the pool of suspects
- so you can investigate knowing which it is - Gram + or gram - are targeted by certain antibiotics
- & some antibiotics target both
If someone has a gram + infection & you give them an antibiotic that targets a gram + bacterium…
the good thing is you leave your gram - bacteria that are good alone
- the drug won’t harm all of the good bacteria - means better toxicity just to what you want so general state of health can be more or less maintained
Gram + general features
- plasma membrane
- THICK peptidoglycan
Gram - general features
- plasma membrane
- THIN peptidoglycan layer
- outer membrane
What is the Gram Stain procedure?
- Apply CRYSTAL VIOLET stain purple & basic:
Gram + = purple
Gram - = purple
Human cell = purple - Apply IODINE = a mordant - intensifies bound stain
Gram + = purple
Gram - = purple
Human cell = purple - Apply ALCOHOL = a decolourizer
Gram + = purple (trapped in cage like structure - alc bulked them up so they can’t get mordant & crystal violet out)
Gram - = colourless
Human cell = colourless (b/c no cell wall) - Apply SAFRANIN (pink & basic) - sticks to any living cell
Gram + = purple (pink will stick but will stay purple b/c it never came off & its darker)
Gram - = pink
Human cell = pink
What are the differential stains?
- The Gram Stain
- Acid fast stain
- Endospore stain
The Gram Stain
- Differential Stains
- Gram positive
- Gram negative
Gram positive
cells that retain a primary stain
• Purple
Gram negative
cells that lose the primary stain
• Take color of counterstain
• Red or pink
Why do gram + not turn pink after step 4?
purple is darker so it will trump
If I gave you a staining procedure, & in that staining procedure it was like a gram stain but everything was mixed up/added diff. things. If in step 1 of this gram staining protocol, inside of crystal violet, she added a yellow basic stain & in step 4 instead of safranin, I added a black basic stain, what will be the outcome for that gram stain result/ For a gram +, - & euk?
everything will be BLACK - b/c you added (+)ly charged stains, but you added the darker one AFTER the alcohol decolourization so it’s gonna stick to gram -‘s just like safranin would have, sticks to gram +’s & is gonna trump yellow b/c it’s darker
Acid fast stain
- differential stains
- Detects mycolic acid in the cell wall of the genus Mycobacterium
- Mycobacterium – retains primary stain
• Fuchsia (pink)
Anything else on slide – color of counterstain
• Blue
Endospore stain
- Endospores retain primary
- Green - malachite green - sticks to endospores if present
- Cells counterstained
- Pink - safranin + - stick to all things that contain a - charge
- Ex. Bacillus anthracis spores.
ONLY produced by SOME bacteria
- always be a gram + bacterium
Mycobacterium genus
has plasma membrane, gram +, & MYCOLIC ACID (hydrophobic) as the outside of their peptidoglycan
Mycolic acid
- hydrophobic
- outside of their peptidoglycan
- unique to members of mycobacterium genus
- can engage with things ALSO hydrophobic
Mycobacterium CANNOT…
undergo a gram stain
- so has to do AFS
Will mycobacterium be able to engage with a basic or an acidic stain?
No - b/c “like dissolve like”
- can engage with things that are also hydrophobic
- acidic/basic carry a charge so it’s hydrophobic
When you gram stain a gram + bacterium, what’s the outcome you expect?
the stain to remain purple b/c it’s trapped, it won’t come out (not outer membrane)
Mycobacterium
retains primary stain
• Fuchsia (pink) - carbol fushin sticks to mycolic acid
Methylene blue (basic & blue)
Anything else on slide – color of counterstain
• Blue - methylene blue (basic & blue)
- basic is +, so blue stain will stick to cells (any cell that’s not acid fast); so other gram +’s, -‘s, human cells that might be in the sample (anything with a net - charge on its outermost surface, methylene blue will try to stick too trying to create a differential output
What are endospores?
resilient structures
ex: if a bacterium finds themselves in a situation where water/nutrients are limited, they can enter in spore state (metabolically inactive) where he hides out until conditions become more fav. for survival, then he comes out & wakes up & starts metabolism & being normal again
ex: rice
- when they fuel replication from those conditions, the microbial count goes up!
- & if you take it & put it on fridge, it will grow slowly
- can destroy with an autoclave- combines temp & pressure for ex
How would you expect a (-) endospore stain to look?
everything would be pink
If everything here was pink (- endospore stain), would you be able to tell me what the result of a gram stain is?
def not - b/c you'll have bugs there that the pink is sticking too but you don't know if its gram + or - (it can be either) - it's only when you know it produces endospores, that you know it must be gram + & therefore will produce a purple stain
If you chose this endospore forming organism, but instead chose to do a gram stain. What would I see as a result of the gram stain on an organism that produces a (+) endospore stain?
purple - b/c it’s an endospore forming & gram + are the only kind of bug that’ll form an endospore & it’s not all
What does the result of this AFS mean (on slide with blue and pink)? How would you interpret the results?
mycobacterium is gonna be there - pink will be members of mycobacterium genus
- BLUE (hydrophilic stuff) is other cell types - everything else that’s there
- (+) result
How would you expect this AFS stain to look if it was a (-) result?
no pink - everything blue (just blue would tell you no mycobacterium, but won’t tell you gram +/-)
Phase-contrast microscopy
Phase ring amplifies differences in the refractive index of cell and surroundings
• Improves the contrast of a sample without the use of a stain
- therefore, doesn’t constitute characteristics of viability
• Allows for the visualization of LIVE SAMPLES
- so you study further or to protect if it’s the last one
• Resulting image is dark cells on a light background
Dark field microscopy
- inverted like how we saw in the previous
• Specimen is illuminated with a hollow cone of light
• Only refracted (bent) light enters the objective
- everything else will go around so it’s not gonna make its way up through the lens & will not be part of the image you get as a result of the eye piece
• Specimen appears as a bright object on a dark background
- Used to observe bacteria that don’t stain well (meaning it’s gonna be hard to visualize)
- Ex) Treponema pallidum – the causative agent of syphilis
Fluorescence microscopy
- Used to visualize specimens that fluoresce
- Emit light of one color when illuminated with another color of light
absorption wavelength: characteristic of the chemical properties of a fluorescent particle
- have to know what this is so it can be absorbed
- have to strike this with light of a particular wavelength
emission wavelength: what’s given off to provide the fluorescents that you see
If absorption wavelength is 450 nm, what can you say about the emission wavelength?
LARGER wavelength - b/c of the energy that was absorbed, some of it was converted to heat - so not all of it will be there
- b/c there is a loss of heat, when this gets absorbed you’re gonna have less that’ll come out which means that wavelength must be larger b/c larger wavelength is lower energy which counts for amount spent in absorption activity
Cells may fluoresce naturally
• Ex. Photosynthetic Cyanobacteria (ORIGIN OF THE EUKARYOTIC CHLOROPHYLL) have chlorophyll
- have photosyn. pigments that have the opportunity to absorb light
- Absorbs light at 430 nm (blue-violet)
- Emits at 670 nm (red)
Or after staining with Fluorescent dye
Ex) DAPI (has affinity for DNA) specifically binds to DNA (allows you to localize it)
- anything in fluorescent blue is an indication of DNA
Immunofluorescence
- Take antibodies (highly specific to 1 antigen)
- HIGH SPECIFICITY of binding to a target - Couple it to a fluorescent particle on the other end
- That fluorescent particle has a certain absorption & emission wavelength
- Take a cell, & apply the antibody, knowing fully well it will go in & bind to a SPECIFIC REGION of the cell
- TREAT cell with the absorption wavelength, & then leave it alone so it emits & you have a detector to pick up on the emission wavelength
Can use a # of antibodies, targeting a # of diff fluorescent particles, in order to allow the opp. to be able to see things inside the cell
BENEFIT: more bang for your buck - b/c you’re not just staining for 1 thing, but for other things as well!
Differential interference contrast (DIC) microscopy
- Uses a polarizer to create two distinct beams of polarized light
- Gives structures such as endospores, vacuoles, and granules a three- dimensional appearance (but still a light microscope - so not amazing but not perfect)
- Structures not visible by bright-field microscopy are sometimes visible by DIC
- can see curvature & dimensions of nucleus
What microscopes improve Contrast in Light Microscopy
- Phase-contrast microscopy
- Dark field microscopy
- Fluorescence microscopy
What microscopes image cells in 3-D?
- Differential interference contrast (DIC) microscopy
2. Confocal scanning laser microscopy (CSLM)
Confocal scanning laser microscopy (CSLM)
• Uses a computerized microscope coupled with a laser source to generate a three- dimensional image (will see layers of the cell b/c of where the laser is engaging)
- when you get that layer, you take it & put it together with another layer of laser image - stack on top of one another)
- building image of the cell
- Computer can focus the laser on single layers of the specimen
- Different layers can then be compiled for a three-dimensional image (can tell a lot of dets)
• Resolution is 0.1 μm for CSLM (μm = light microscope)
- not clearest ever
μm =
LIGHT microscope
e- microscopes =
nm ranges of resolution
- smaller than light microscopes
Electron microscopes
use electrons instead of photons to image cells and structures
• Wavelength of electrons is much shorter than light (therefore higher E) –> higher resolution
BETTER UPPER LIMIT OF MAGNIFICATION
- also even if you HOLD magnification constant the resolution beats a light microscope!
Two types of electron microscopes:
- Transmission electron microscopes (TEM)
* Scanning electron microscopes (SEM)
Transmission Electron Microscope
- Electron beam focused on specimen by a condenser
- Magnets used as lenses
- Electrons that pass through the specimen are focused by two sets of lenses
- Compound microscope (combining 2 things that are happening)
• Electrons strike a fluorescent viewing screen (providing the image)
- magnet is condenser & inverted same way as light microscope
- High magnification and resolution (0.2 nm) (nm = 10^-9m)
- Specimen must be very thin (20 – 60 nm)
*stained adhered differently based on the characteristics of those structures, which means that now the image that’s created as the e-‘s are deflected back to the viewing screen is gonna be heavily detailed
Unstained cells do a poor job of scattering electrons
- won’t be able to see anything - b/c e-‘s have poor penetrin
• Must be stained with (heavy) metals (means sebitonic particle # is high & so e- # is high, creates e- density that allows you to visualize when e- beam strikes the sample)–> lead or uranium
- therefore heavy metal stain is gonna be e- dense
- stick to thin section diff. on a ribosome then it would stick to a DNA region/membrane
• Bind to cell structures to make them more electron dense
• Enables visualization of structures at molecular level
- can see inside of cell (not alive not) but lot of work
THINK: egg - has to cut to visualize the inside
- slice to see diff. dets
- BUT cell is 60-80% water
- think: cutting water balloon
- so must permeate it with a resin - resin is injected as a liquid to polymerize & harden once it goes inside
- then all internal materials of that cell are fixed & isolated to 1 particular region
- then use a microtome = diamond knife to do thin sectioning
- see diff. in texture, see morphology of nucleoid region where chromosome located, see characteristics of mem. cross-section & all fine dets on surface on cell
Microtone
= diamond knife to do thin sectioning
When to use a Transmission Electron Microscopy (TEM)
when you wanna see INSIDE of cell
Light vs. Electron microscope (450x)
e- microscope:
- way better than light
- even when magn. is constant
- great solution
therefore, even when magn. is set constant, the e- microscope provides such a great clarity b/c resolving power is gonna be much smaller than the light microscope
Scanning Electron Microscopy (SEM)
• Specimen is coated with a thin film of heavy metal - e- dense! (e.g., 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 specimen’s SURFACE.
- clarity
- can see surface dets (round, amount of space, arrangement, contour)
- good job at providing external dets
Scanning Electron Microscopy (SEM) process
e-‘s sprayed on an angle on the surface
- thin coat of heavy metal adhering to the side
e- beam slowly scans
- provides it’s blunt at bottom, narrow at top, out like this on side
- fine surface dets
When to use a Scanning Electron Microscopy (SEM)
when you want to see OUTSIDE of cell (contour)
Prokaryotes (before nucleus)
- bachelor suite (everything happens in the open - no separate compartments, everything within boundaries of plasma membrane)
• NO membrane bound nucleus or organelles
- genetic material floating
• Generally smaller than eukaryotes
- pack a lot less
- Simple internal structure
- Divide by binary fission (EQUIVALENT OT MITOSIS)
• Most are unicellular
- if they’re multi they will not have tissue differentiation (just will be power in #’s)
How is binary fission in prok’s equivalent to mitosis?
- such that in the absence of any error during DNA replication, this process produces genetically identical daughter cells, so you don’t expect any genetic variation - it’s meant solely as a means of increasing cell #
- diff. when we do mitosis, we get a total cell # that’s higher
- but when BF is completed they get a whole new organism b/c they are unicellular
Bacteria (Eubacteria)
true bacteria
• Diverse metabolism
- allows them to explore so many of the diff. environments that exist on the face of the earth
- means ton of diversity & a vital role in every ecosystem found (creating imp. characteristics - if removed, it’ll be impactful)
• Live in a broad range of ecosystems
• Pathogens (cause disease) and non-pathogens (cannot cause disease)
- a lot of change can potentially occur - not consistent all throughout life (genetically plastic)
Archaea (Archaebacteria)
NOT BACTERIA - only thing they share with bacteria is their prok. cell structure
- Diverse metabolism
- Live in extreme environments
- that allow them opp. to thrive, b/c can live under low pH conditions sometimes & increased salt
- but can also live in mild ones (like our gut)
- some can live @ 30% NaCl (& still can maintain water nonetheless), our blood is 0.9%
• Non-pathogens
- has capacity to change
- through picking something up (a weapon)
- via a gene set that can be produced into a toxin or capsule that can lend ability to cause disease
Cell Morphology
- -> shape!
- diff. in morphology have nothing to do with intelligential ability, height, weight, interpret lang, etc.
- as well as nothing to do with genetics of cell, comparisons & contrasts of other species, just a unique characteristic to fully understand the organism
- physical characteristic totally unrelated to almost all other characteristics
- shape gives a bit of a push to a direction/hint of the cause based on how it looks to diagnose an infection
Cell Morphology types
- Coccus (pl. cocci)
- Bacillus (pl. bacilli)
- Spirillum (pl. spirilla)
- Cells with unusual shapes
- Budding & appendaged bacteria
- Filamentous bacteria
Coccus (pl. cocci)
• Roughly spherical
• Ex) Streptococcus pyogenes
- causes strep throat/flesh eating disease (organisms are not the same strain)
- like “tall” in name
Bacillus (pl. bacilli)
- Rod shaped (asymmetrical shape)
* Ex) E. coli
Spirillum (pl. spirilla)
- Spiral shaped
* Ex) Spirillum volutans
Cells with unusual shapes
• Spirochete
- LONGER in length, allows them to bend (FLEXIBLE)
• Ex) Treponema pallidum
- cause disease of syphilis
Budding & appendaged bacteria
appendaged - allows opp. to make contact with the surface (attachment is imp.)
- appendage to allow opp. to bind to a particular structure
- stick to surface to not get flushed away
- we are flushing clean (vomit, urinate, defectate, etc.), these so we need to have an organism that can grab on if it has capacity to stick when it gets deposited to inside of body, otherwise all these self-cleaning mech’s are gonna eject organism from where it came from
Ex) Caulobacter crescentus
Filamentous bacteria
Ex) Streptomyces griseus
- produces imp. antibiotics
- increase SA for things like absoprtion
- b/c long & thin
- better opp. to take nutrients in
Morphology typically does not…
Morphology typically does not predict physiology, ecology, phylogeny, etc. of a prokaryotic cell
Cell Morphology may be…
selective forces involved in setting the morphology
What are the selective forces that may be involved in setting the morphology?
• Optimization for nutrient uptake (small cells and those with high surface-to- volume ratio)
- SA:V will do better
- b/c they have better/more membrane to interact with their environ., allowing the opp. for the cell to protect itself against environ. changes
- nutrients come in, get distributed & cell is benefiting from that
• Swimming motility in viscous environments or near surfaces (helical or spiral-shaped cells)
- if a cell has a particular shape its able to cut a lot of that material so its able to disseminate & relocate to other locations
• Gliding motility (filamentous bacteria)
- filamentous structures have opp. to glid over the surface (move from A –> B in order to max. contact with environ., use physical support as a means of locomotion)
- use physical support as a means of locomotion rather than free-living movement
Describe cocci & the different types
cooci - individual cell structures
- can choose to arrange themselves in more complex patterns
- -if they choose to get together in a more high level organization, they are still individualized cells that live autonomously
- still indiv. cells that are free living but chose to get together
- diplococcus (diplococci)
- diplobaccillus (diplobacilli)
- streptococcus (streptococci)
- staphylococcus (staphylocci)
Streptococcus (streptococci)
doesn’t have anything to do with the name but ppl chose to include morphological dets & arrangements within the name/genus name (strepto)
If you took a throat swab from a person who has streptococcal infection (strept throat) & you looked at it under the microscope & saw what would you be thinking in terms of the causative agent of that person’s pharyngitis?
strept throat - whereas if you didn’t see that or if you saw a baccilis, autonomically you’d be realizing its something else
Mono
manifests almost same as strept throat, doctors confuse/misdiagnose
- virus WOULDN’T grow freely b/c it’s not a free living cell, so you wouldn’t see anything if you took a throat swab to try to identify
- therefore, wouldn’t see infection agent b/c virus’ can’t be cultured that way (would just see normal flora)
Why would the cocci WAIT until they have power in #’s to do something bad?
a lot easier to kill one
- then they will start transcribing/translating a toxin or something
Prokaryote Sizes
- Average
• E.coli (asymmetrical) ~ 1.0x3.0μm
• Staphylococcus aureus ~ 1.0 μm diameter - Very small
• Mycoplasma (as genus more have cell wall) genitalium ~ 0.3 μm - exception
- respon. for sexually transmitted infection (STI)
- anomaly b/c it does NOT have a cell wall –> outermost component = plasma membrane (sounds like an ANIMAL cell b/c no cell wall)
- also small b/c they’ve come to rely on the host for many things
- extremely efficient - pack lighter
- pathogenic & really tiny so when they enter body they can enter tight tissue
- Very large
• Epulopiscium fishelsonii ~ 80 x 600 μm.
Mycoplasma (as genus more have cell wall) genitalium ~ 0.3 μm
- very small
- exception (as genus none have cell wall)
- respon. for sexually transmitted infection (STI)
- anomaly b/c it does NOT have a cell wall –> outermost component = plasma membrane (sounds like an ANIMAL cell b/c no cell wall)
- also small b/c they’ve come to rely on the host for many things
- extremely efficient - pack lighter
- pathogenic & really tiny so when they enter body they can enter tight tissue
Would you put Mycoplasma genitalium (very small prokaryote) in a gram +/- designation or not?
How would they look if you did a gram stain & they were part of the sample?
No
Appear pink - look like a Euk
- b/c alc would destain their membrane & then go on & stain pink by the end
- doesn’t mean gram - either, but just the way all organisms without a cell wall will appear
- so gram staining not effective for identifying this b/c it will look like any gram - cell
Cell Size and the Significance of Being Small
Surface-to-volume ratios, growth rates, and evolution
• Advantages to being small
• Small cells have more surface area relative to cell volume than large cells (i.e., higher S/V)
• Support greater nutrient exchange per unit cell volume
- distributes nutrients & have less to make everytime you have to make a new cell (b/c small & don’t have to make organelles)
• Tend to grow faster than larger cells
- v. efficient
- turn over cell a lot faster
Significance of Being Small
Large cell V = 1 - membrane you're able to interact with your envir. with to bring nutrients in etc. SA:V ↓↓↓: 1 ex) 2
Individual (small) cells V = 1 - small, therefore tons of opp. to bring nutrients in & share those nutrients with entire volume on inside of cell SA:V ↑↑↑:1 ex) 10 5x greater
Lower limits of cell size
- you wanna be small & pack light but you can only get so light before you can’t bring your essentials with you
• Cellular organisms <0.15 μm in diameter are unlikely (b/c can’t fit dets req. for survival (ribosomes ex)
- anything smaller they can’t fit key dets
- Open oceans tend to contain small cells (0.2–0.4 μm in diameter)
- Many pathogenic bacteria are also small (missing many genes whose functions are supplied to them by host)
- rips out pages of recipe book they don’t use anymore b/c they grow it in garden
- able to whizzle way in tissue (has to be small)
What are key points of the Membrane Structure (Cytoplasmic membrane (cell or plasma membrane))?
• THIN structure that surrounds the cell
- thinner it is, the easier it will be for nutrients to be able to transit into cell & wastes to exit cell
- Vital BARRIER that SEPARATES cytoplasm from environment
- Highly SELECTIVE PERMEABLE BARRIER (can chose what goes in/out (control); enables concentration of specific metabolites and excretion of waste products
- metabolites - necessary for metabolism & fuel generation (ex: ability to let glucose in)
- waste products - imp. b/c can be toxic if accumulated
What is a key defining characteristic of a cell? & why is this not one for viruses?
CYTOPLASMIC MEMBRANE (cell or plasma membrane) is the key defining characteristic of a LIVING cell - prok, euks
BUT except viruses - b/c no plasma membrane, therefore no living features to be able to move things in/out & control things imp. for life
What is the composition of membranes?
- General structure is PHOSPHOLIPID BILAYER
- Contain both HYDROPHOBIC (fatty acid) and HYDROPHILIC (glycerol-phosphate) components
- Can exist in many different chemical forms as a result of variation in the groups attached to the GLYCEROL BACKBONE
- Fatty acids point INWARD to form hydrophobic environment; hydrophilic portions remain EXPOSED to external environment or the cytoplasm
Amphipathic
phospholipid bilayer is this b/c it has a head group that prefers interactions with water (hydrophilic) & tails that prefer interaction away from water (hydrophobic)
Describe the glycerol backbone & attachments
has a head group that DETERMINES IDENTITY & also TAIL LENGTH & UNSATURATION that can also determine characteristics/behav. of the structure
ex: longer tails –> more sturdy
unsat. creates more fluidity
How is the phospholipid bilayer based on the Hydrophobic Effect?
phospholipids naturally come together in water in order to promote fav. interaction & circularize (won’t be living in water)
- therefore, won’t be exposed b/c their touching water which is undesired but they become an uniform structure
Imagine a beaker with oil and you put PL’s in. What will they do in terms of the motivation forming a higher level structure?
Will stick head groups to inside so they have fav. int with each other & tails pointing out getting fav. int. with oil (“hydrophobic like hydrophobic”)
- think: inverse micelle
Ester phospholipids consist of:
- 2 Fatty acids
- Phosphate
- Side chain (optional)
Ester
CH2-C=0
|
O
|
C
carbonyl group attached
- impacts heat stability & beh.
Amphipathic
has both polar and non-polar characteristics
- arranged to meet demands of both ends of molecule
Polar
molecule carries full or partial charge
• Hydrophillic
Non-polar
molecule is uncharged (CH2 - no charge - therefore non-polar)
• Hydrophobic
What can we say about the bonds b/t N & H in +NH3?
e-‘s held closer to N b/c polar covalent bond
- therefore, partial +
Cytoplasmic membrane
- 8–10 nm wide
- Embedded proteins
- Stabilized by HYDROGEN BONDS and HYDROPHOBIC interactions
- Mg2+ and Ca2+ help STABILIZE membrane by forming ionic bonds with negative charges on the phospholipids
- SOMEWHAT fluid
What particularly will be determining width of membrane?
LENGTH OF FA CHAINS
- will determine how far apart head groups will be from 1 another
- if you adjusted the FA tail length (bigger or smaller), the protein wouldn’t have portions that are hydrophobic contacting tails properly & portions that are hydrophilic contacting the tails properly
- critical to have tail length how it should be otherwise like & like won’t be interacting as it should!
What’s the net charge on a membrane?
(-)
How do membranes stay together so nicely if they have a compounded net (-) charge?
DIVALENT CATIONS (Mg2+ & Ca2+) - bring (+)ity to bond & alleviate repulsion - stabilizes (-)ity so membrane don't have strong forces (repel) forcing it apart
How is the larger structure held together in an organized way in the Cytoplasmic membrane? In the f.a tail region
WdW’s
- weak int. b/t non-polar group
- still take heat to break
What interactions stabilize the 4 points on the protein and b/t the head groups in a Cytoplasmic membrane?
H BONDS & IONIC BONDS
- respons. for creating a higher level structure in these regions
Explain how the Cytoplasmic membrane is somewhat fluid
VdWs require heat to break
- monounsat tails allows VdW’s int. that req. heat to break
- since tail is far apart - you are not forming VdW’s there so it creates a FLUIDITY
- imp. to be somewhat fluid for optimal function
Membrane proteins
• Outer surface of cytoplasmic membrane faces the environment
• In gram-negative bacteria, interacts with a variety of proteins (periplasmic proteins) that bind substrates or process large molecules for transport
- meaning diff. proteins floating in periplasmic space & outer surface will interact with those proteins
- Inner surface of cytoplasmic membrane interacts with proteins involved in ENERGY-YIELDING REACTIONS and other important cellular functions
- Integral membrane proteins
- Peripheral membrane proteins
Integral membrane proteins
Firmly embedded in the membrane
can be transmembrane integral or just integral
Peripheral membrane proteins
One portion anchored in the membrane
- usually attached to EC face of mem. or IC face attached to hydrophilic regions, usually H+ bonds & ionic int. (electrostatic int. b/t (+) & (-)ly charged areas)
- to stay fixed
Which proteins will you expect to find on the INNER surface of the plasma membrane of a bacterium, that’s involved in the production of energy?
on inner mitochondrial membrane is ETC
- the inner surface of cytoplasmic membrane is analogous to inner mitochondrial membrane which will have ETC present within
Archea prefer…
EXTREME enviromental conditions (therefore, must have cell structure able to tolerate)
____ linkages in phospholipids of Archaea
ETHER
- O holding together
- better thermal stability
- stronger
- able to survive more environmental extremes
Bacteria and Eukarya that have ____ linkages in phospholipids
ESTER
- carboxylic acid
Archaeal membranes
- Archaeal lipids LACK fatty acids; have isoprenes instead
- Major lipids are glycerol diethers and tetraethers
- Can exist as lipid monolayers, bilayers, or mixture
Isoprenes
in Archaeal membranes
C5 tails
hydrophobic & come together
C5 + C5 = C10
C10 + C5 = C15
Glycerol diether
- glycerol structure that has a phosphate as part of its head group
- 2 hydrophobic tails attached to glycerol by just O (2 ether linkages)
bilayer
in Archaeal membranes
Diglycerol tetraethers
- 2 glycerols
- tails are connecting them
- forms a mono layer - NO gap b/t 2 tail structures
- will get more VdWs
- which means we have more that we’ll have to break to be able to melt
- @ higher temps. the membrane will be able to hold its shape better with more VdWs
Archaeal membranes can also have a mixture for their lipid layer
depending on how many of each you have - it’ll determine at a partic. temp if you’re solid, semi-fluid or liq
- membrane ALWAYS must be semi-fluid
- mixture will have lil bit of fluidity & also lil bit of stability
What Archaea membrane would they want @ 4 degrees celsius?
want gaps in b/t - lipid bilayer to prevent solidification
What Archaea membrane would they want @ 60 degrees celsius?
no gaps in b/t - lipid monolayer - a lot of stability to prevent melting
Lipid bilayer vs. Lipid monolayer
Lipid bilayer:
- gap
- no VdW’s
- separated –> less thermal stability
Lipid monolayer:
- 1 layer - no gaps
In contrast to lipid bilayers, lipid monolayer membranes are…
extremely heat resistant
↑↑ VdWs
Lipid monolayer membranes are extremely heat resistant. So where are they commonly found?
Commonly found in hyperthermophilic Archaea (grow best at temperatures above 80oC)
- Archea that perfers extrem. or excessively high temps
- increasing # of monolayers, having more VdW’s, allows them to be semi-fluid @ those temps
What is able to pass through the lipid bilayer?
small/non-polar molecules from [high] to [low]
What is able to pass through the lipid bilayer VIA A PORE?
*polar molecules are more controlled/need transporters/channels
pores are highly specific, polar material can move
Membrane Function
• PERMEABILITY BARRIER
- Polar and charged molecules must be transported
- Transport proteins accumulate solutes AGAINST the concentration gradient (active transport)
(ex: glc transport does this from low to high concen., therefore where it isn’t there that part of the mem. is non-permeable to that solute so unable to so you can concen. it against its gradient)
PROTEIN ANCHOR
• Holds transport proteins in place
- to be in the right location for function
ENERGY CONSERVATION
• Generation of proton motive force
Do the proteins 1-4 have to stay in that position, or can they move?
MUST stay in that order (anchored) or else they will lose function of ETC
How do protons move then?
can move protons to outside of cell thorugh complexes 1,3 & 4 - building a [ ] gradient, where they can flow back in to allow for ATP synthase
- producing energy
If proteins were able to flow wherever they wanted b/c charge didn’t prevent that from happening, what would be the issue? (high H+ outside, didn’t have to use ATP syn)
GRADIENT WOULD DISSIPATE (energy stored within would be lost - not harvested)
- protons have to stay there & then flow in - controlled
Cyanide (toxin) makes a membrane leaky to proton. Explain
Makes a membrane leaky to proton.
Able to move from high to low concen. (don’t need to use ATP syn).
Lose ability to harvest that energy & ability to produce ATP & drive Na+/K+/ATPase to keep gradient from neurons firing.
Becomes lethal - energy prod. goes to a hault & brain runs out of fas
Carrier-mediated transport systems
- Show saturation effect
* Highly specific
Simple diffusion
molecule is moving directly through the plasma membrane (moving along [ ] gradient)
- no transporter/transmembrane protein
- inefficient
Why is simple diffusion inefficient?
b/c inside of mem. is not really hospitable to that movement (wait a long time for it to come in)
- can use a transporter to help
Transporter
carrier; physically binding to, & then guiding a molecule into a cell to let it go
Explain the effect of a transmembrane protein(carrier)/transporter/transporter saturation
polar inside
- get dramatic increase in solute entry & then levels off when all binding sites are saturated inside of carrier protein, it can’t go any faster in terms of internalization
- saturation effect
- & is highly specific-bringing in 1 particular molecule
Three major classes of transport systems in prokaryotes
- Simple transport
- Group translocation
- ABC system
What do all 3 major classes of transport systems in prokaryotes require?
All require energy in some form, usually proton motive force or ATP
- ALL ACTIVE!
Simple transport:
driven by the energy in the proton motive force
*SECONDARY ACTIVE TRANSPORT - energy released from PROTON movement from [high] to [low] funds energy needed to move another SOLUTE from [low] to [high]
Group translocation:
chemical modification of the transported substance driven by phosphoenolpyruvate
- solute will STOP moving into cell @ equilibrium
- wants to EXCEED equil. since they’re in an envir. with abundance of nutrients (wants to bring more in past equil.) b/c it’s not sure if in a hour or so there won’t be any nutrients
- by MODIFYING it when they bring more in
How does solute exceed equil (bring more in past equil.) when equil. is reached?
by MODIFYING it when they bring more in
- modification comes with phosphorylation
- once modified, it’s not the same molecule again (think: hat on head (changed - not same)
- so won’t reach an equil. b/t molecule & molecule phosphorylated (diff.)
- outcome is that conc. of solute inside the cell, consistently stays low, therefore you can bring in even more (maximizes your intake potential)
ABC system:
periplasmic binding proteins are involved & energy comes from ATP
- PRIMARY active transport
- ATP binding domain on inside of cell that allows transporter to take ATP & hydrolyze it; releasing energy
- energy is then used to pay for solute that moves across the mem. to inside of cell
Three transport events are possible:
uniport, symport, and antiport
Uniporters
transport in one direction across the membrane
Symporters
function as co-transporters
Antiporters
transport a molecule across the membrane while simultaneously transporting another molecule in the opposite direction
- cotransport - move 2 solutes with same transporter
Protons ALWAYS HIGHER ______ celll
OUTSIDE
Describe E. coli ex with a symporter
Lac permease is bringing H+ into cell (releasing energy b/c high to low) & then the lactose is active (going against - req. energy)
- proton is being used to pay for movement of the lactose
- nutrients that have been taken up are the lactose
Simple transport: Lac permease of Escherichia coli
- Lactose is transported into E.coli by the simple transporter lac permease, a same direction
- Activity of lac permease is energy-driven (to pay for it)
- Transports lactose and a H+ into the cell simultaneously
Group Translocation - e.g. phosphotransferase system in E. coli
(takes up glucose)
- Sugar is phosphorylated during transport across the membrane
- Moves glucose, fructose, and mannose
- Phosphoenolpyruvate (PEP) donates a P to a phosphorelay system
- P is transferred through a series of carrier proteins and deposited onto the sugar as it is brought into the cell
- normally energy release here is used to form ATP by substrate level phosphorylation
- but here, you take phosphate group & use energy from that rxn to transfer it through those enzymes & then drops it on glucose that’s coming through
- PE-P is the most energy rich molecule that exists (breaking it releases energy that is used for substrate level phosphorylation - to produce an ATP molecule)
- trying to est. equil., the outcome is it will eventually equalize, putting a stop to glucose uptake by phosphorylating it
- becomes Glucose 6-P (no longer glucose - therefore no longer part of glc gradient & so glc can continue to come in)
ABC (ATP-binding cassette) transport systems
• Involved in uptake of ORGANIC compounds (e.g., sugars, amino acids), INORGANIC nutrients (e.g., sulfate, phosphate), and trace metals
- BRINGING IN VITAL THINGS for cell
- Typically display HIGH substrate SPECIFICITY
- Gram-NEGATIVES employ periplasmic-binding proteins and ATP-driven transport proteins
- Gram-POSITIVES employ substrate-binding lipoproteins (anchored to external surface of cell membrane) and ATP-driven transport proteins
Gram-_______ employ periplasmic-binding proteins and ATP-driven transport proteins
negatives
Gram-_______ employ substrate-binding lipoproteins (anchored to external surface of cell membrane) and ATP-driven transport proteins
positives
What does, “typically display high substrate specificity” mean for ABC transport?
- NEED A LOT, v. imp. nutrient uptake & life
- specific - separate one for each molecule
- huge part of genome is dedicated to providing instructions to make these diff. ABC transporters b/c you need a separate 1 for each nutrient & protein info in order to assemble the structure originates from genes within the DNA
Porin non-specific transmembrane protein
molecules can pass through, as long as they are polar & have the appro. size characteristics, the molecules are able to pass through
- think: fence
Periplasmic binding protein
specific to nutrient
ABC Transport is…
highly specific
Describe ABC transport systems in a Gram - cell
- PORIN in OUTER mem. that’s NON-SPECIFIC as long as you meet size criteria & polarity, you can go through
- Wandering around as a nutrient in PERIPLASMIC SPACE that’s large & endless
- PERIPLASMIC BINDING PROTEIN (SPECIFIC) for good guys-nutrients to that nutrient & deliver to the ABC transporter (HIGHLY SPECIFIC) which will allow the nutrient to be taken to the inside of the cell
Describe ABC transport systems in a Gram + cell
- Reaching around, swishing around in ECF
- Finds nutrient for which its specific (substrate binding lipoprotein - specific)
- Brings to door & guides it through ABC transporter (highly specific)
(no outer mem. to create boundary & periplasmic space)
ABC transporters (ATP-binding cassette)
Solute binding protein
• Periplasm
• Binds specific substrate
Integral membrane proteins (transporter)
ATP-hydrolyzing proteins
• Supply energy for the transport event (costs 2 ATP)
Cell Walls of Bacteria and Archaea
Outside the cell membrane
- Rigid –> Helps determine cell shape (regardless of how mem. might appear underneath - will see the outermost strong layer)
- NOT a major permeability barrier (as long as the molecule is small enough (unrestricted) it can bypass that layer
- Porous to most SMALL molecules (think: jungle gym net - anything that can fit through; provided that it’s the right size (small) - will go through that layer
- Protects the cell from osmotic changes
The cell wall of Bacteria and Archaea are, “Not a major permeability barrier,” & “Porous to most small molecules.” How is this imp. for designing antibiotics?
imp. for designing antibiotics that go into a cell to target a ribosome & inhibit protein syn., antibiotic must have size characteristics that allow it to go through the cell
______ is a unique feature for every single living cell
plasma membrane
Plasma membrane =
our skin - our guaranteed outermost layer
Describe an ex of a hypertonic cell in a hypotonic solution
water will rush in by osmosis [low] to [high] b/c of the hyper/hypo
- plasma mem. will swell (bigger) b/c of additional water
- & cell wall pushes back exerting a counter pressure but the cell won’t burst b/c the cell wall protects from osmotic changes
In terms of cell wall:
- Cell wall won’t be able to provide indefinit protection (can only protect so much)
- if cell continues to swell b/c of continuous influx of water
- outcome is it will rupture - Cell wants this added layer of protection b/c anything that happens to this 1 cell could be the end of the organism as a whole –> highly protective
- ECF & cytoplasm has 0.9% [NaCl]
- but bacterium can find itself anywhere so constantly will have alternate external environment
- so need to be able to adopt or prevent serious changes to cell structure since it is not constant like ours is
Explain if a deflated balloon is in a glass and then air goes in
balloon = plasma membrane
air - makes bigger until glass exerts counter pressure on that expanded balloon
glass = cell wall
if you keep adding more air in, the glass is not gonna help & the outcome is the balloon will blow
What does penicillin actively interfere with?
Penicillin actively interferes with formation of peptidoglycan
- peptidoglycan on an organism that’s growing doesn’t have cross links (not strong)
- a bacterium trying to grow in presence of penicillin, will have holes blown in side of its plasma membrane b/c cell burst without a strong cell wall there to protect against osmolarity issues
Function of the cell wall
• Cell wall prevents cell expansion – protects against osmotic lysis
• Protects against toxic substances – LARGE hydrophobic molecules (CAN’T fit through pores therefore unable to get through - outcome: cell is protected against chemical threats in this way)
Ex) detergents, antibiotics
• Pathogenicity (ability to be able to escape immune detection or immune capture - able to get away from immune system & the destruction of its own self)
- Helps evade host immune system (imp. for survival)
- Helps bacterium stick to surfaces (better chance for bacterium to persist inside the body - since body is trying to constantly flush clean)
• Partly (b/c some might also have a cytoskeleton) responsible for cell shape (cocci, bacillum, spirilum). (b/c its a rigid structure - even if mem. has dehydrated away - the cell wall maintains external shape)
Describe isotonic, hypotonic & hypertonic
Isotonic: no net movement of water
Hypotonic: water moves into the cell & may cause the cell to burst if the wall is weak or damaged (osmotic lysis)
- not living anymore (internal contents leak out)
Hypertonic: water moves out of cell, (b/c water always moves to more hypertonic envir.) causing its cytoplasm to shrink (plasmolysis)
- cytoplasm pulls back from cell wall
- not dead, just dehydrated
- can rehydrate & become metabolically active
Describe why honey doesn’t need to be refrigerated
- doesn’t need to be refrigerated
- it’s so hypertonic that bacteria in there are dehydrated (therefore, impossible to spoil)
- not enough water to metabolize sugar & grow and increase in #
- can store at RT b/c maintains semi-fluid viscous material
Cell wall is…
anything outside of plasma membrane
