BoC MT practicals

Question 1

How do you set up a microscope to view a sample under bright field conditions ?

Answer 1

Ensure sample in light beam, adjust light
Lowest power objective lens in place, move sample as close as possible to objective lens then lower until focused (focus moves stage height)
Set condenser iris to mid position, gradually close until image of a field iris appears (hexagon)
Adjust condenser up/ down, sharpest image of hexagon
Centre hexagon using screws-moves condenser and ensures focused beam of light hits sample, centres field iris (hexagon+field iris)
Open field iris until hexagon disappears-> whole filed of view illuminated with bright/even light.
Remove eyepiece, condenser iris fully opened, then reduced to 2/3 of whole field ( x10, x40) opened as far as possible for x100

Question 2

What are the 3 lenses of a microscope and what does each do ?

Answer 2

Eyepiece- focuses image onto eye, inverts
Objective- responsible for magnification of image of sample
Condenser-Focuses light on the sample

Question 3

What are the purposes of the condenser iris and field iris?

Answer 3

Field iris- controls the area of illumination
Condenser iris-controls diameter of beam of light produced by condenser lens, occurs before lens- controls diameter of light reaching lens

Question 4

What special arrangements are required for using the 100x objective?

Answer 4

Oil lens, requires immersion oil between slide and lens to function
Oil placed when nose piece in empty position
Set up as normal but hexagon will never look as clear, point at which condenser focuses is more blurry
Condenser iris opened as large as possible , not just to 2/3 of diameter

Question 5

What is the actual spatial resolving power of a microscope?

Answer 5

d-minimum resolved distance in micrometres 
numerical aperture (NA)- range of angles from which lens can accept light 
d= 0.61x wavelength of light in micrometres x numerical aperture

Question 6

What is cytoplasmic streaming?

Answer 6

Observed in Chara (algae)
Myosin moves along actin filaments that cover surface of chloroplasts/organelles(visualised using TEM), drags organelles and entrains cytoplasm
Overcomes limits of cellular diffusion for large cells, distributes metabolites, substrates organelles.

Question 7

How are the slides of Chara cells prepared?

Answer 7

4 dots of grease on slide support cover slip , drop of pondwater added and small branch of chara, coverslip added and pressed down gently.

Question 8

Why does the focus need to be adjusted when viewing the chara cells?

Answer 8

Thickness of Chara cells is greater than the depth of focus of the microscope , focus is adjusted to build up 3D image of cell

Question 9

How is cytoplasmic streaming measured in the cells? Why might the times measured differ?

Answer 9

Graticle, time taken for object to move set distance along graticle measured , larger objects in the cytoplasm move more slowly. Most of moving objects are clumps of nuclei , chara are multinucleate cells

Question 10

Where does cytoplasmic streaming occur inn the cell ? In what direction ? What is the indifferent zone ?

Answer 10

Chara cella have large vacuole, then layer of cytoplasm with lots of parallel rows of chloroplasts. Vacuole at some points connects with cell wall/membrane ->indifferent zone with no chloroplasts/free cytoplasm for cytoplasmic streaming.
Cytoplasmic streaming occurs with spiral flow, with flow occurring in opposite directions on either side of the indifferent zone. Flow reverses at tips of chara, no vacuole. Rotational and bidirectional flow.

Question 11

How could the roles of actin and myosin be tested in cytoplasmic streaming?

Answer 11

Tested by differentially fluorescently tagging actin and myosin , observing cells under microscope using fluorescent light

Question 12

What are the different types of microscope? How do they compare?

Answer 12

Bright field,Phase contrast(both light)
Fluorescence
Transmission and scanning (electron)
light microscopes-limited resolution on 0.2 micrometres
electron- resolution of 1nm, can be used to observe intracellular structure

Question 13

What are the differences between scanning and transmission electron microscopy? What does the resolution depend on ?

Answer 13

Resolution depends on -wavelength of electron beam, acceleration/ velocity of beam
Transmission-1nm reso
works in a vacuum , specimens treated so differing structures transmit electrons with differing efficiency. Electrons focused onto phosphorescent screen/CCD. Electromagnets focus beam/ magnify
Scanning-10nm reso
Specimens scanned with beam of electrons and secondary electrons emitted collected to form an image, visualisation of 3D surfaces of structures

Question 14

What are the main staining methods used?

Answer 14

Negative staining-heavy metal salt stain, strongly electron scattering, forms opaque background, specimens stand out light against, good for small structures
Thin section-Cells fixed, macromolecules cross linked by chemical treatment
Dehydrated by soaking in ethanol
embedded in epoxy resin, sections cut using diamond knife, stained using heavy metal salts(TEM) <100nm thin
Freeze fracture- rapidly frozen to -180, cut with cold knife-> fractures sample
Fracture lines influenced by strucure of specimen. Coated in platinum, strengthened with carbon. Platinum layer floats off, used .(TEM) membrane structure
Coating with golf-
Increases secondary electron production and prevents charging->SEM

Question 15

How are the pancreas cells prepared? Why ?

Answer 15

Fixed with glutaraldehyde and stained with toluidine blue , ensures good preservation of cellular structure.
Binds to proteins based in charge, shows general cell strucure

Question 16

What is the basic structure of the pancreas section?Acinar cell function/form?

Answer 16

Consists of densely packed with thin tubes, acini,one cell thick, composed of acinar cells. Cut all in different planes , may be difficult to interpret.
Enclose central lumen of each acinus
Acinar cells produce digestive enzymes in the form of zymogens , zymogen granules stained deep blue , all located in apical region ready for secretion into lumen of acinus, triggered by arrival of food in digestive tract.
All other organelles in basal region

Question 17

What does the methyl green and pyronin stain show? What does this highlight about the cell?

Answer 17

Methyl green-DNA stain
Pyronin-stains RNA pink
Doesn’t give good general cell structure but shows distribution of DNA and RNA in cell
DNA in nucleus in basal region of cells
RNA distributed throughout cell, but particularly concentrated at basal region-> endoplasmic reticulum located mainly in basal region of cell, extension of nuclear membrane
Structure of typical of cell involved in protein secretion-ribosomes/ER throughout cell but concentrated around nucleus

Question 18

Why is an RNase added? What does it demonstrate?

How else can RNA distribution be observed?

Answer 18

RNase added to pyronin stained cells , demonstrates stain binds to pyronin as only nucleus remains pink, RNase cant diffuse over nuclear double membrane.
Can also be observed by fluorescently tagging RNA, either by tagging uracil or by using a probe for a sequence universal to all mRNA

Question 19

How can an amoeba be observed? What are it’s main features?

Answer 19

May need to use high brightness , fine focus used to observe all features( thickness is greater than depth of focus of microscope) Have extensions, pseudopodia, used for movement through extension and retraction , cytoskeleton.Central nucleus, food vacuole, contractile vacuole-periodically evacuates then grows, controls water content. Ectoplasm, portion of cytoplasm on pseudopodia devoid of granules.

Question 20

What is phase contrast microscopy?

Answer 20

Enhances contrast in transparent/colourless objects by influencing optical pathway of light. Element in condenser must be aligned with element in phase contrast lens
PH1-X10
PH2-x40
PH3-x100
Generally requires more light

Question 21

What is fluorescence microscopy ? How do fluorescent markers work?

Answer 21

Visualises molecules/structures using fluorescent markers
Fluorescent markers absorb one wavelength of light , emit longer wavelength-> excitation and emission spectrum. Fluorescent component of molecule-> fluorophore. Light passes through excitation filter, only certain wavelengths pass, focuses onto sample by a dichroic mirror , emitted light passes through emission filter
Can detect low numbers of fluorophores and be used in live cells
Resolution limited, light microscope(200nm , potentially 50nm), requires fluorescent tags, can disrupt function of molecules being followed
Multicolour imaging- multiple probes with different colours of fluorophore used, must have non overlapping emission spectra

Question 22

How are the specimens made to fluoresce ?

Answer 22

• > direct, fluorescent molecules like chlorophyll
• > covalent labelling with fluorophore
• > Gene insertion of GFP into proteins being observed, recombinant gene
• > Immunofluorescence- primary antibody for specific macromolecule added, then secondary antibody carrying fluorophore added

Question 23

What is confocal microscopy?

Answer 23

Catches light from a single plane of focus, improves image quality, uses pinholes
Can cause light loss, v intense light has to be used -> lasers. Can only illuminates 1 point as a time so sample scanned sequentially and digital image produced. High contrast, high resolution image. Improves fluorescence microscopy

Question 24

How can molecules be directly quantified using spectrophotometers?

Answer 24

Molecules which absorb light, can be measured directly, amount of light absorbed directly proportional to molecule concentration. Often molecules have delocalised electron systems(NADH, ds/ssDNA , RNA, chlorophyll) proteins with lots of aromatic amino acids
Calculated using beers law
Absorbance at given wavelength= conc in moles/l x molar extinction coefficient litres/cm/moles x path length of cuvette cm
For nucleotides generally mg instead of moles- length of molecules more important than numbers

Question 25

How can molecules which don’t absorb light be quantified? What external factors as well as concentration can effect the light absorbed?

Answer 25

Can be measured using assay- Bradford assay, used for proteins, based on coomassie dye, changes colour when it binds to basic amino acids.
Bound to basic amino acids- deprotonated form is most stable , blue SO3- group, 595nm
Acidic environment, protonated form most stable, red, 465nm
Presence of detergent, affects protein folding-> affects which bases exposed for bonding
Composition of protein (how many basic aa)
Beers law CANNOT be used, relationship between absorbance and concentration is not linear at higher concentrations-> standard curve produced

Question 26

How is the protein standard curve constructed ?

Answer 26

BSA protein, phosphate buffered saline-> different protein concentrations, vortexed to mix. Bradford reagent added, vortexed, given some time to bind but not enough to settle
Spectrophotometer calibrated using phosphate buffered saline and bradford reagent, even unbound bradford reagent may cause some absorbance, must be quantified.

Question 27

How is soluble protein extracted from tobacco leaves ?

Answer 27

Plants grown in high/ low light conditions and amount of protein in leaves (mostly photosynthetic apparatus) compared. Cork borer used to remove identical replicate discs of leaf=> roughly same volume of leaf used
Large veins in leaf avoided
Crushed using pestle and mortar, in PBS solution, proteins released from cells-> solution
Crushed until homogenous to ensure proteins extracted from the whole disc
Stored on ice to prevent degradation by proteases released from vesicles within the cell upon crushing , don’t function/ function so so slowly at such low temperatures.
Centrifuged to separate out soluble protein, centrifuge MUST be balanced.
Bradford reagent added, protein standard curve used to estimate protein content, assumes BSA is good assay for plant cellular protein ,has similar aa composition and number of exposed basic aa

Question 28

What are the key formula for this practical?

Answer 28

Beers law
For converting concentration to concentration
v1xc1=v2xc2
Same amount
Amount to diluted amount
n1/v1=n2/v2
Always ensure you check units when using rules

Question 29

How can protein structure be determined?

Answer 29

X-ray crystallography- requires crystals
NMR spectroscopy- atomic structure of proteins in solution, requires highly concentrated solutions, can only do small proteins. Measures inter proton distance , protein dynamic as in cell
Cryo EM-sample rapidly frozen, then observed, can have high resolution but can’t be used on small proteins

Question 30

Details of an alpha helix?

Answer 30

HB form with groups 4 residues ahead, rise per residue is 0.15nm, HB length-0.267-0.291nm . All residues project outwards as no space in centre. Amphipathic helix, often membrane spanning, residues distributed so all hydrophobic residues are on one side while all polar residues are on the other. Hydrophobic interactions often form between chains, polar residues face each other forming hydrophilic pore. If predominantly hydrophobic , likely transmembrane as cytoplasm-> water

Question 31

Details of beta pleated sheet?

Answer 31

HB -0.52nm, rise per residue 0.37-0.38nm, chains/sheets held together by HB.
General-interior of protein molecules often hydrophobic , chains held together by hydrophobic interactions.

Question 32

Where are disulfide bridges formed ?

Answer 32

Disulfide bridges, only form key part of cytosolic structure for non cytosolic proteins, cytosol is reducing environment. In chloroplasts/mitochondria-> oxidising environments

Question 33

Acidic/basic amino acid charge at physiological pH, effect on acidic residues being in hydrophobic pockets

Answer 33

At physiological pH-basic aa positively charged at pH 7
acidic aa negatively charged at pH 7
Acidic residues surrounded by hydrophobic pockets-deprotonation is less likely, charged residues in hydrophobic pockets unfavourable, raises effective pKa of residues gives up hydrogen ion less readily

Question 34

What is the Ramachandran plot? Where will the dots form?

Answer 34

Ramachandran plot , plots the phi (n-c) and psi(c-c) angles of secondary structures(restricted rotation due to resonance hybrid of peptide bond. Have characteristic angles for secondary structures, larger the protein is , more dots deviating from characteristic areas due to links, B reverse turns etc

Question 35

What is the structure of aquaporin 1 and how does it relate to its function?

Answer 35

Transmembrane water channel, homotetramer 4x alpha helices, 6 transmembrane, 2 internal
269 residues, narrow pore-prevents entry of hydrated ions, lined with hydrophobic residues-permit rapid water transport
Outward facing polar residues at top and bottom of alpha helix , mostly composed of outward facing hydrophobic residues
Ramachandran plot , points mainly in lower left portion - alpha helix, but some also in upper left portion, reverse beta turns, loops etc

Question 36

What does lysozyme do? How can it be assayed?

Can beers law be used ?

Answer 36

Digests bacterial peptidoglycan by hydrolysing polysaccharide backbone. Bacterial cells lyse.
Small enzyme, tough. M luteus- gram positive bacteria (much more affected by lysozyme than gram negative, don’t have outer membrane), suspension scatters light, destruction of bacteria by lysozyme reduces light scattering-> reduces apparent absorbance in spectrophotometer (measures turbidity of suspension). Light scattering doesn’t fully obey beers law, not true absorbance, but changes in attenuence reasonable measure of enzyme activity.

Question 37

What is the procedure for creating a calibration curve?

Answer 37

Swirl suspension (or will settle out), sodium phosphate buffer used , has appropriate pH for functioning of enzyme. Spectrophotometer calibrated using cuvette of sodium phosphate.
Different concentrations of M luteus used , volume must be kept constant at 3ml, must be well mixed to ensure even distribution of M.luteus,  vary the water content of the cuvette, amount of sodium phosphate buffer MUST be kept constant  to ensure pH is constant and amount of light scattering due to buffer is constant.
Calibration curve produced.

Question 38

What is the procedure for assaying lysozyme activity

Answer 38

Control experiment- M.luteus and phosphate buffer added , placed in spectrophotometer, take reading of attenuance and ensure it doesn’t change with time
Add lysozyme, mix, measure attenuance every 10 seconds for 2 mins . Repeat (check reproducibility)-mean values
Repeat with different volumes of lysozyme, buffer added / removed to keep volume constant at 3ml (may be bad experimental design, would change ph/light scattering de to buffer, see above)
Initial rate, 10 to 30 s after mixing

Decrease in attenuance with time- exponential, catalytic rate of each enzyme constant but amount of M.luteus decreases. Exponential character shallow but increases as amount of lysozyme added increased
Initial rate, linear function of amount of enzyme added, substrate initially in excess
Although at high concentrations, this is less accurate.

Question 39

What is the effect of pH on enzyme activity? Why ?

Answer 39

Activity of lysozyme depends on 2 residues, Asp-52 and Glut-35. Glut-35 is in a hydrophobic microenvironment, is protonated at physiological pH as hydrophobic environment increases effective pKa(6.0) . Asp-52 is deprotonated (pKa=4.5). Changing pH can affect this protonation as it changes local environment of residues. Enzyme functions best at pH above 5.8 and below 7.2. As pH increases, though pH change buffered by microenvironment, glu-35 residues can become deprotonated, lysozyme no longer functions
pKa, relative strength of acid. PH=Pka+ log([A-]/[HA]) , Pka=pH when equal amounts in protonated and deprotonated forms, lower pka-stronger acid.

Question 40

What is the purpose of enzyme cofactors? NADH as a cofactor.

Answer 40

Cofactors help enzyme enlargen functionality beyond that of natural amino acid side chains
NADH , very common cofactor in redox enzymes, nicotinamide segment responsible for redox activity, delocalised ring structure which can shift

Question 41

Why can NADH be used to assay enzyme activity ? What are the 2 types of assay?

Answer 41

NADH and NAD+ have different absorption spectra, NADH absorbs much more 340nm light than NAD+.
Assays can be continuous- assay giving continuous reading of product, discontinuous- samples taken periodically, reactions stopped and conc of substrate/ product determined

Question 42

Why might the absorbance of NADH over time not be entirely linear?

Answer 42

Product inhibition- systematic deviation, product is structurally similar to substrate , proportion of product doesn’t leave active site.
Enzyme gets less active- after number of turnovers can be seen , could be due to denaturation by partial unfolding
Falling substrate conc- can decrease velocity of reaction , only when substrate conc falls below km value

Question 43

How can Km be determined? For which enzymes?

What is Kcat?

Answer 43

Km can be determined using Michaelis menten plot, plots initial reaction velocity against substrate concentration. Km is the substrate concentration at half of vmax- represents rate of degradation of enzyme/substrate complex over rate of formation.
Lineweaver burke plot provides much more accurate estimation , 1/michaelis menten equation, gradient=km/vmax, intercept is 1/vmax.
Kcat =Vmax/[E]

Question 44

How are chloroplasts extracted from lettuce leaves?

Answer 44

lettuce leaves blotted dry, central veins removed, healthy green tissue is homogenised with ice cold sucrose medium adjusted to appropriate pH( sucrose-isotonic with stroma, ice cold, prevents enzymes released by homogenisation from degrading proteins). Muslin wetted using sucrose medium, muslin-> funnel, medium filtered through funnel , squeezed into beaker in ice bucket.
Poured immediately into centrifuge tube,cold, chloroplasts sedimented, supernatant discarded. Sucrose medium added to resuspend chloroplasts until homogenous, kept on ice in the dark- prevents O2 being generated

Question 45

How is chlorophyll content measured?

Answer 45

sample of chloroplasts treated with acetone, disrupts membrane and releases chlorophyll . Centrifuged to remove precipitated protein, balance centrifuge. Photometer zeroed with 1ml of acetone, then measure absorbance with 1ml of sample. Absorbance value multiplied by 2.9, gives mg/ml of chlorophyll in original sample.

Question 46

How does an oxygen electrode work ?

How is the oxygen electrode calibrated ?

Answer 46

Oxygen generated moves through teflon membrane (only permeable to oxygen), undergoes reduction reaction on platinum cathode generates H2O2 , results in generation of current proportional to amount of O2 in the system
Anode made of silver
Calibrated by adding sodium dithionite crystals to water when stirrer on, removes all dissolved 02. Do NOT put lid on electrode during this process. Signal reading should fall to 0. Reaction vessel washed out multiple times with distilled water , removes all sodium dithionate, remove washes using pipette, don’t scratch teflon membrane. Shake RO water, fully saturate with
oxygen, calibrate to 1000(top end ) while stirrer is on (magnetic flea will be moving). Readings can be converted from mV to O2 concentration using the fact O2 solubility in air saturated water is 0.255 micromole /ml ,placing 1.5ml in chamber

Question 47

How is the electrode used to measure photosynthesis and deduce effect of herbicide ? Why is KCN/ferricyanide added ?

Answer 47

sodium phosphate buffer(optimum pH) K3Fe(CN)6 and KCN added. Buffer leads to bursting of chloroplasts, NADP and electron acceptors diffuse away from membrane fragments must be replaced by artificial acceptor, ferricyanide accepts electrons from PSI and PSII. KCN inhibits cytochrome oxidase of mitochondrial respiratory chain, prevents O2 usage. Mixture bubbled through with nitrogen to mix. Chloroplasts then added, chamber lid put on, collar region placed on and lid screwed down, ensure no air bubbles and that meniscus is halfway up capillary tube, check at eye level-> smallest possible area of contact with atmosphere.
Stirrer on , cover electrode with bag (should be no photosynthesis) , take readings every 20s for 2 minutes, then remove bag , take readings, add herbicide , take readings until rate is stable.
Repeat with phenylquinone, accepts electrons only from PSII, ensure light constant distance from oxygen electrode.

Question 48

How is photosynthetic rate calculated in this experiment ? What is the conclusion of this experiment?

Answer 48

Rate measured in light -rate measured in dark (photosynthesis doesn’t occur in dark , measurement must be due to something else) Ignore first measurement taken after light first switched on, more accurate calculation.
herbicide targets PSII, rate of oxygen evolution decreased in both cases, any inhibitors acting after PSII would have no effect on oxygen evolution