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