Cell biology

name the two types of light microscopes

compound microscopes and stereo microscopes

describe the difference between compound and stereo microscopes

Compound microscope relies on light passing through the specimen

Stereo microscopy is great for dissecting small subjects, however stereo is not great for imaging cells in a tissue

What type of microscope is able to see very small molecules and atoms?

Electron microscope

describe resolution

Resolution means how far apart two objects need to be to be able to see them separately. A shorter wavelength allows us to see things with a greater resolution. 

Describe what about electron microscopes allow them to view smaller things with a greater resolution

Electron microscopes uses high energy electrons (Gamma rays) which have a much shorter wavelength (<0.01 nm) and higher frequency compared to photons which are used in a light microscope, allowing a greater resolution

What are requirements/ setbacks of using electron microscope?

Requires sample to be in vacuum

Samples must be dehydrated

Act of imaging damages subject

Only gives monochrome images

What are the two forms of electron microscopes?

Scanning (SEM) and Transmission (TEM)

what are the differences between SEM and TEM

SEM:
• Detector at an angle to stage
• Detects scattered electrons
• Gives ‘3D’ images (xref stereo)
• No need for sectioning
TEM
• Electrons pass through sample to
detector
• Gives ‘flat images’

Describe fixing

Fixing preserves the specimen
• E.g. soak in formaldehyde/glutaraldehyde
• Embed and dehydrate in resin/paraffin

Describe sectioning

Sectioning (microtome) takes a thin slice
of the specimen

Describe staining

Staining sections enhances contrast
• Dyes stain different cellular components
• Heavy metals (eg uranyl acetate) for EM

Describe the membrane of a eukaryotic cell

Made up of a bilayer of phospholipids (Amphipathic lipids) , Hydrophobic core, separating aqueous compartments

Describe the polarity of the phospholipid

Hydrophilic head, hydrophobic fatty acid tails 

What can your membrane contain?

phospholipid bilayer,  Cholesterol, glycoproteins, glycolipids

How are membranes dynamic?

 They can undergo fission and fusion (dividing into two/ combining to form one)

What does fluorescence microscopy allow us to do?

Allow us to see things in high resolution with high detail. 

What is the relationship between the energy of a wavelength and its colour

Blue light has more energy than red light

Blue (High energy) Red (Low energy)

define flouraphore

a chemical compound that absorbs light energy at a specific wavelength and re-emits light at a longer wavelength.

If a cell appears red, what colours would it absorb and reflect?

Absorb blue light, reflect red light

What happens when a molecule absorbs light

When a molecule absorbs light electrons gain energy and when this happens its excited and moves further from the nucleus

The excited electron will release this energy and will emit a photon and return back to its relaxed/ground state

What does the difference in energy between the ground state and the excited state reprisent?

The difference in energy between the ground state and the excited state is equivalent to the colour that molecule will look at, however the electron will lose thermal energy therefore some energy is lost and the photon emitted will be closer to a red colour.

What does fluorescence do?

Fluorescent methods use

different excitation and

emission wavelengths

Makes them very sensitive

Can be quantified accurately

Wavelengths are controlled using filters

Illuminates sample with a specific

colour of light (excitation)

Detects a higher wavelength of

light (emission)

What colour does DAPI stain?

Blue

What colour does Propidium Iodide (PI) stain?

Stains DNA red but can only enter dead cells

What colour does Acridine orange stain?

DNA green

RNA orange

What is the purpose of immuno fluorescence 

Is able to identify specific areas of proteins

How does immuno fluorescence work?

Fluorophore stains conjugated to an antibody The antibody’s specificity localises the stain to particular cell components

What is a downside of immuno fluorescence

takes a long time

What is the purpose of green fluorescent protein?

Is encoded by a gene so we can use genetics to add this gene into organisms to produce a green fluorescent organism. 

It was originally found in a jellyfish

The GFP gene can be fused with a target gene to study gene expression.

What is the purpose of Confocal Microscopy?

Can take visual sections of extremely thin focal plane  through a specimen allows 3D reconstruction.

What does FRAP stand for ?

Flourescence Recovery After Photobleaching

How does FRAP work

It allows us to determine the speed that a lipid molecule can move through the membrane, as it burns through the organelle leaving only the membrane and then over time lipid which is also stained will return into the organelle and we can measure the time it takes for the fluorescence to return.

What does FRET stand for?

Forster Resonance Energy Transfer

What does FACS stand for

Fluorescence Activated Cell Sorting

How does FACS work?

Fluid flow creates a stream of single cells

Size and fluorescence measured of each cell Cells separated into droplets and charge applied

Droplets separated by charge

What is FACS useful for?

• Classifying immune cells

• Isolating stem cells

• Assessing cell viability

• Finding rare cancer cells

What are bacteria

simplest organisms

What are the shapes of bacteria?

Rod- shaped (Bacillus), Spherical (Coccus), spiral (Spirillium)

What type of cell are bacteria?

Prokaryotic

What are charecteristics of Prokaryotic cells

No nucleus - DNA in a nucleoid
• Inner membrane surrounded by
peptidoglycan cell wall in the
periplasm.
• Gram-negative bacteria have an
additional outer membrane.
• Capsule of polysaccharide (sugars)
• Flagella for motility
• Protein fibres – pili, fibrils, fimbriae

Label this prokaryotic cell

IM = inner membrane
PP = periplasm
PG = peptidoglycan cell wall
OM = outermembrane
C = capsule (sugar coat)
N = nucleoid
P = plasmid
R = Ribosome
F = flagellum
Pi = pilus

Describe eukarytoic microbes

Have a nucleus (with nuclear
membrane, pores and endoplasmic
reticulum)
• Mitochondria (endosymbiotic
bacteria)
• Fungi (including yeasts)
• Protists (everything eukaryotic which
isn’t plant, animal or fungus:
• Algae (plant-like protists)
• Protozoa (animal-like protists)
• Fungus-like protists

Describe characteristcs yeast (Fungi cells)

• Small (5 um) and simple eukaryote
• Contain the subcellular structures
common to all eukaryotic cells
• Chitin cell wall (like cellulose with N)
• Can divide like bacteria by binary
fission (fission yeast –
Schizosaccharomyces pombe)
• Some bud (budding yeast,
brewer’s/baker’s yeast –
Saccharomyces cerevisiae)
• Other fungi have complex life cycles

Describe charavteristics of protists (Eg. green algae)

• Photosynthetic (chloroplasts –
endosymbiotic bacteria)
• Can be flagellated (Euglena,
Chlamydomonas)
• Diatoms grow a silica shell (frustule)

What are the foour main types of Protists - Protozoa

Four main types (polyphyletic):
• Mastigophorans/Flagellates – move with flagella
• Sarcodines/Amoebae
• Apicomplexans – immobile parasites
• Ciliates – move with cilia

Describe cillia

• Cilia are essentially the
same across the
eukaryotes, including
those lining our lungs.
• Unlike flagella, they are
enclosed within the cell’s
plasma membrane
• Dynein is a ‘motor’ protein
which powers the flexing
of fibers within the cilium

Describe the cell boundry of ciliates

• An inner membrane under the plasma
membrane
• Alveolar spaces for strength
• An underlying cell cytoskeleton or ‘pellicle’

What is DNA composed of

DNA is composed of individual nucleotides

What are the three main compnents of a nucleotide

Phosphate group deoxyribose sugar, nucleobase

Describe the order of organisation within a chromasome

Phosphate group deoxyribose sugar, nucleobase make up a nucleotide. many nucleotides make up a strand of DNA and two strands hydrogen bond together to form a double helix of DNA. DNA then wraps around histones tightly forming chromatin fibres which are then coiled further to form a chromasome

Where is the 5’ end and the 3’ end on a nucleotide?

5’ is on the phosphate group and the 3’ end is on the empty hydroxyl group.

How are individual nucleotides bonded together?

Condensation reaction to form a Phosphodiester bond

(A phosphodiester linkage forms between the 3’ C of the sugar in thymine and the P of the phosphate group in guanine. Water is lost so it’s a condensation reaction)

How many hydrogen bonds do A and T/U form

2

How many hydrogen bonds do C and G form

3

What are the differences between DNA and RNA

RNA has uracil instead of thymine

On C2, Ribose has a hydroxyl (–OH) group

Uracil has a hydrogen (–H)

DNA has thymine instead of uracil

Deoxyribose has a hydrogen (–H).

thymine has a methyl (–CH3) group.

How does DNA stores the genetic code

Transcription

How does DNA transfer the code into RNA

Translation

What are the products of transcription and translation?

proteins that carry out the functions of the cell

What phase of the cell cyle does DNA replication occur?

S phase

What enzymes does DNA replication require

Topoisomerase and helicase

DNA polymerase and ligase

Topoisomerase and helicase

Unwind the DNA helix

What does DNA polymerase and ligase do?

 join individual nucleotides and synthesise new DNA strands.

What name is given to the way DNA replicates itself

Semi-conservative

( One strand of the parental DNA is conserved in each of the new molecules formed.)

What are the two strands in DNA named during semiconservative replication?

Leading and lagging 

What direction does DNA polymerase add new nucleotides

in the 5’ to 3’ direction only.

Describe replication in the leading strand

On the leading strand DNA polymerase is moving towards the replication fork so add nucleotides to the 3’ end of the new strand and can copy continuously

What is the name of the enzyme that unzips DNA to form a replication fork?

DNA helicase

What is the name of the enzyme that joins individual nucleotides to synthesise a new DNA strand?

 DNA polymerase

At which end of DNA molecules are new nucleotides added?

5’ end toward the 3’ end

What is the difference between the way leading and lagging stands of DNA are synthesised?

 Lagging strand must be replicated in short fragments called okazaki fragments, multiple primers and DNA polymerase are uses

 What is the name of the enzyme that makes the lagging strand complete?

DNA ligase