Practicals Flashcards
Resolution an magnification of different microscope types?
Light: 0.2um 100x
TEM: 1nm 10,000,000x
SEM: 10nm
How should specimins be prepared for a TEM
- Fixed cells
- Dehydrated
- Embedded in plastic resin
- Sliced into thin sections (100nm)
- Stain using heavy metal salts
What is negative staining and when is it used?
Useful to visulise small structures such a protein molecules, virus particles and microtubules
- Use heavy metal stain (strong electron scattering ability)
- Specimins will stand out as light objects
How to prepare specimin for a SEM?
Coat specimin with conductive material such as gold
Useful to get detailed pictures of intact specimins
What is the function of the condensor?
Focus’s light onto the specimin
The condensor assembly sits directly beneath the stage
What are the irus’s of a microscope?
Two iris’s: the field iris and condenser iris
The two iris’s must be aligned with the optic axis and adjusted for optimal viewing of the specimin
What is the turret disc and when is it adjusted?
Part of the condenser assembly: turns to alternate between phase contrast and bright field viewing
What is the function of the condensor focus?
Raises/lowers the whole consenser assembly
What is the function of the focus?
Moves the stage
Setting up a microscope for bright field microscopy
- Insert specimin
- Focus the specimin using the fine and corse focus
- Bincoular tube adjustment (distance between eyepieces)
- Focus and centre the condensor
- Adjust consensor iris
How to focus and centre the condensor?
Use the lever that controls the consensor iris to the middle position
Slowly close the field iris gradually until hexagon comes into view
Use condensor focus to sharpen the image of the hexagon
Centre the hexagon using condensor centring screws
Open the field iris until the edge of the hexagon just disappears
How to adjust the consensor iris?
Remove one of the eyepieces and look down the baralell
Shift the consensor iris lever completly to the left hand side to fully open consensor iris
Close the condensor iris until the diameter of the illuminated circle is 2/3 of the whole field
Return the eyepiece
What do do when changing to 40x?
Only use fine focus to focus the specimin
Focus and centre condensor as before
Field iris adjustment as before
Consensor iris adjustment as before
What to do using 100x magnification?
Use immersion oil and gently raise the stage stage with corse focus
What is the spacial resolving power of a microscope?
Smallest seperation at which two objects can be distinguished
d = 0.61(theta)/NA
d = minimum resolved distance (um)
theta = light wavelength
NA = numerical apature of objective lens
What are the 4 adjustments you should make to the microscope each time you change lens/specimin?
- Condensor focus (hexagon)
- Condensor centreing
- Field iris diameter (open until hexagon just disappears)
- Condensor iris diameter (2/3 of whole field)
When is bright field microscopy used?
Viewing structures which absorb some of the incident light
-*stained slides or pigmented cells**
But many cells have little absorption
When is phase contrast microscopy useful and why?
Examining specimins such as biological tissues that do not absrob a lot of light.
It enchances contrast in transparant tissues, allowing us to see structures in cells
Stains for DNA and RNA?
DNA: Methyl green stains DNA green/blue
RNA: Pyronin stains RNA pink
What do methyl green and pyronin stain?
Methyl green stains DNA green/blue
Pyronin stains RNA pink
What does the stain toludine blue stain?
Zymogen granules in acini of kidney blue
How is set up of phase contrast achived?
Rotate turret disc (moves different optical elements into the light path)
Generally, more light is need, so increase the brightness
What are pseudopodia?
The lobes of ameoba that extend as they move
How may cells be fixed for TEM viewing?
Cryofixation and chemical fixation
What is freeze fracture and its use?
Method of preparing specimin for TEM
1. Rapidly freeze sample in liquid nitrogen
2. Fractured with cold knife
Useful to look at internal structure of cells and membranes
Negative staining: what and use?
Useful for looking at very small particles
Specimin suspended in heavy metal salt
3 ways to prepare a specimin for TEM
- Thin specimin
- Freeze fracture
- Negative staining
Use of florecnse microscopy?
Provides specific infomation about the location of particlar proteins/molecules within a cell
How does a flourcent molecule work?
Absorbs light at one wavelength and emits it at another - detectable at very low concentrations
Ways to use flouresnce microscopy?
- Naturally flourecent molecules in living cells
- Flourecent dyes:
- bind to specific organelles or molcules or ions - Immunoflourecnse
- Fixed cells are permeabilised with detergent
- Antibodies move into the cell and attach to complimentry proteins
- Secondary antibody binds to primary antibody and is fixed to flourcent dye - Flourecent proteins
- GFP
- Use recombinant DNA technology to join them to other protein encoding genes
Why might laser scanning confocal microscopy be used?
Out of focus light from the ordinary floursent microscope is removed
How does the bradford assay work?
Based on coomassine dye
Changes colour from red to blue when it binds to basic amino acids (mainly arginine)
Limitations of bradford assay
Relies on binding of coomassine dye to basic amino acids (less basic amino acids - less strength)
Equation relating amount and volume for diluted samples
n1/v1 = n2/v2
Equation relating concentration and volume
V1C1 = V2C2
What can be measured to determine the action of lysozyme on a gram positive bacteria?
Drop in apparent absorbance (attenuance)
Because the light scattering ability of bacterial cell walls decreases when damaged with lysozyme
Why is lysozyme such a stable enzyme
Can surive at quite high temperatures (up to 80)
Very small enzyme
Has 4 S-S bridges
What does DTT do to lysozymes?
Denatures by breaking S-S bonds
A strong reducing egent
What is SDS
A detergent that binds to proteins and gives them a strong negative charge
Why do only proteins outside of cells have S-S bonds?
The inside of cells has reductive power which would break the disuphide bonds (thioredoxin)
How to tell if a hericide is targeting PSII or PSI?
Phenyol quinone only accepts electrons from PSII
If rate of oxygen evolution is decreased with the addition of phenyol qunione, the herbicide must target before PSII
Ferricyanide can accept electrons from both PSII and PSI.
If rate of oxygen evolution only falls after binding with Ferricyanide, but not phenyol qunione, it must target the electron transport chain in between PSII and PSI.
Mating process of saccchormyces cerevisiae
May be alpha or a mating types (each secrete a different pheremone)
When two types come close together, they arrest at same point in the cell cycle
Morpholgical channeces fuse the two haploid cells to create a diploid cell (schmoos)
Diploid cells divide by budding until starvation conditions (no N source or fermentable sugar)
Starvation conditions drives entry into meiosis and sporulation
4 haploid spores produces (a tetrad)
Life cycle of aspergillus fungi
Grow as lomg chains of cells (hypae)
When food supply runs out, asexual spores are produced
These spores will germinate to form a myeclium in the right conditions (asexual life cycle)
Sexual life cycle
- Two strains fuse and form a mycelium (heterokaryon)
- If the heterokaryon is oxygen deprived, gametic formation takes place
- Meiosis produces haploid asospore, which form a fruiting body that will germinate to form hypae under the right conditions (asexual life cycle resumed)
What is a heterokaryon?
Fusion of two fungi
Multinucleated with two types of nuclei
How can heterokaryons be used to test if two independent mutations affect the same gene/metabolic function?
If the two strains cannot grow apart by but the heterokaryon formed by fusion can, the mutations must be on two different genes (complimentation)
Why is bakers yeast useful for studying mitochondrial inheritance?
Can survive the loss of mitochondrial DNA
Faculative anarobes
- Individuals with mutations in mtDNA can grow on glucose but not on non-fermentable carbon stores
- If they have mutations and grown on glucose, they form smaller colonies (petites)
How are mitochondria of yeast uniparentally inherited>
Mutant mitochondria segregate through mitosis during vegetative divisions
A diploid cell that has gone through several mitotic divisions contains only one type of mitochondrial genome
Why can petite diploid not produce spores?
Meiosis and sporulation relies on mitochondrial function, so while they can survive as haploids, they cannot surive as siploids
What is partial digestion of DNA
When a restriction digest is stopped before reaching completition, some molecules are not cut at all of their sites and extra bands will appear on the gel.
How to test if a mutation in a biosynthetic pathway is present on just one gene or mutiple
Create replica plates of yeast colonies, each of which is deficient in a different intermediate of the biosynthetic pathway
If the mutation is only in one gene, all colonies would be able to grow on all plates but one
If it is in mutiple genes, there is multiple plates that they would not be able to grow on
Features of a good primer
GC clamps to increase binding efficentcy
Specific/no common sequences to prevent mispriming
Avoid repeated runs of GGGGGG
No complimentry regions between primer pairs
How can mispriming be alleviated
Dimethylsulphoxide: DMSO
- changes interactions between primer and template DNA
Change primer design
Hot start - reduces the liklihood of non-specific primer interactions. Keeps the DNA polymerase inactive ensures that primers have properly annealed before synthesis begins.
Reverse complimentation to make primers from DNA??
Remember that DNA synthesises in 5’ - 3’ direction.
Therefore need to check if you need to reverse compliment the DNA to make the primer
Example of flourecent dye to visualise DNA?
DAPI
Binds to AT rich regions with blue flourence
Disadvantages of immunoflourence
Requires fixed cells -
How to tell the anterior/posterior and dorsal/vental of fruit fly embryo
Posterior: pole cells
Dorsal: flatter
Stage of development where germ layers form?
Gastrulation
How to form a ramachan plot?
Psi on y axis
Phi on x axis
3 Hydrophobic Amino Acids
GAV
Glycine - H
Alanine - CH3
Valine - CH(CH3)(CH3)
3 Hydrophillic Amino Acids
STG
Serine - CH2OH
Tyrosine - CH2 + Phenol
Glutamine - CH2CH2C=O(NH2)
What is the indifference zone in chara?
Constriction of the cytoplasm and a seperation of the cytoplasm
There is inversion of cytoplasmic streaming around the indifferent zone.
What is the function of zygomen granules
Contain protien for secretion into th elumen of the acinus
Units for molarity
Mol/L
Dalton definition
1 dalton = 1 g/mol
Molecular mass is measured in daltons
What does the lineweaver burk plot show?
Gradient: Km/Vmax
Y-intercept: 1/Vmax
What is a katal?
1 mole of substrate turned over per second
Should enzyme concentration be high or low for calculaing Km
Lower than substrate concentration
Good model organism
Short generation time
Easy maintenence
Well chracteriested genome
Well characterised mutant stocks
Easily manipulatable
High reproductive rate
Observable phenotypes
Preparing a fixed sample for flouresence microsopy
Fixation + Permebalisation to allow the dye/antibody to enter the cell
What is used to stain DNA in a gel electrophorisis?
Ethidium bromide. Inserts itself between stacked bases of DNA.
Can be visualised under UV light.
How to prepare pancreas cells for viewing their nucleic acids?
100% ethanol agitate
70% ethanol and agitate
Distilled water and agitate
Add methyl green/pyronin stain for 30 mines
Rinse and blot
Add mounting medium and leave to dry
How to prepare cheek cells for viewing under light microscope
HBSS+
Scape inside of cheek gently with a cocktail stick and transfer to solution
Stain with methyl green that turns DNA green/blue
Phase contrast
How to prepare amobea for viewing
Take a slide and place 2 PVC pieces of tape across it to prevent the cover slip squashing the ameboa
Draw amobea into pipette
Drop between the tape and lower coverslip back on
View under phase contrast
How to prepare chara slide
Add four dots of grease slightly smaller than the coverslip
Add some water betwen them
Cut off a group of growing branchlets
Place in the pond water
Gently lower cover clip
View under bright field
Super secondary structures with just beta strands
B hairpin
B meander
Greek key
Describe drosophillia oogenesis
Visualised using immunoflourence
Each ovary has 18 ovarioles
Oocytes progress through 14 stages
Begins in the germarian at the tip of the ovary (posterior)
Germline stem cell asymetrically divides to form cytoblast and a stem cell
Cytoblasts undergo 4 mitotic divisions to form 16 cell cyst (unfinished so cells are joined by a ring canal) - one of these 16 becomes the oocycte
Nurse cells synthesis and transfer RNA, proteins and organelles to the oocyte
Follicale cells form a protective layer
Mature oocyte arrests in metaphase I
What is the synaptonemal complex
Can be tagged using immunoflouresence
Begin to assemble in 4 of the cells in the cyst, then two (proto-oocytes)
Forms during prophase I and facillitates the alignment and pairing of homologous chromosomes
Double strand breaks occur in the SC that allows recombination
Where in mitosis have budded yeast cells arrested?
Before anaphase
