Microscopy Flashcards
1
Q
Key terms: Magnification
A
Magnification is the number of times larger an image appears to be compared to the actual specimen which enables further detail to be seen. Magnification is limited by the resolution of the microscope.
2
Q
Total magnification of the specimen
A
total magnification of the specimen=eye piece magnification (x10) x objective magnification (x4,x10,x40)
3
Q
Key terms: Resolution
A
Resolution is the ability to distinguish between 2 separate points (determines the clarity of the image)
4
Q
Why does an electron microscope have higher resolution?
A
EM have higher resolution as they use a beam of electrons which has shorter wavelength.
5
Q
Why do we preserve specimens?
A
-enables them to be cut into sections to observe under a microscope
-enables them to be treated with a variety of different stains to distinguish between different types of cells, living and dead cells and different chemicals or metabolic processes.
-enables different structures to be revealed
6
Q
Function of eye piece on light microscope
A
-magnifies specimen
-can be dismantled to insert an eye piece graticule to enable specimens to be measured accurately
7
Q
Function of barrel on light microscope
A
-passes light from objective lens to eyepiece
8
Q
Function of turret
A
-holds objective lens
-rotates to enable selection of objective lens
8
Q
Function of objective lens
A
-magnifies and resolves specimen
9
Q
Function of stage
A
-supports slide in correct position at 90 degrees to illuminate source
-enables light to pass through specimen
10
Q
Function of condenser
A
-focuses light from illuminator source onto specimen
11
Q
Function of iris diaphragm
A
-controls the level of light reaching specimen
-best definition achieved with lower light intensity
12
Q
function of substage illumination
A
-source of illumination
-blue light bulb can be used to use light of shorter wavelength to improve resolution
13
Q
How to prepare a temporary slide for observing using a light microscope?
A
-fixation: use 70% alcohol
-staining: use few drops of appropriate differential stain
-mounting: cover with cover slip to exclude dust and air
14
Q
Advantages of preparing a temporary slide
A
-rapid and simple procedure (no complex apparatus or skill required)
-can mount specimen in glycerine to prolong examination period
15
Q
How to prepare a permanent slide for observing under a light microscope? 1
A
1) Fixation
-preserves specimen in lifelike condition
-minimises distortion
-chemicals such as alcohol or acetic acid are added to make proteins and nucleic acids insoluble, fixing them in position
16
Q
Preparation of permanent slide 2
A
2) Dehydration
-removes traces of water from fixed material
-achieved by placing the specimen in increasing concentration of alcohol
17
Q
Preparation of permanent slide 3
A
3) Clearing
-addition of xylol removes dehydrating alcohol
-ensures material is made transparent
18
Q
Preparation of a permanent slide 4
A
4) Embedding
-supports material so it is firm enough for sectioning
-can be resin, plastic or wax
19
Q
Preparation of a permanent slide 5
A
5) Sectioning
-use a microtome to cut fine slices embedded specimen
so that light can pass through the specimen
20
Q
Preparation of a permanent slide 6 7
A
6) Differential staining
-improves contrast between different tissues and structures
-can be permanent or temporary
7) Mounting
-embeds and protects material
ensures material can be observed over a long period of time
21
Q
Iodine-KI solution
A
-for plant specimen
-temporary stain
-colour is blue-black
-used in starch
22
Q
Aniline sulfate
A
-for plant specimen
-temporary stain
-yellow colour
-used in lignin
23
Q
Toluidine
A
-plant specimen
permanent stain
red/purple
lignin and cellulose
23
Eosin
-for plant specimen
permanent stain
red colour
used in cellulose
24
Methylene blue
-animal specimen
-permanent stain
-blue colour
-used in Nuclei reticulocytes
24
Leishman's stain
-animal specimen
permanent stain
red pink blue
red blood cells, nucleus of white blood cells
24
Haematoxylin
-animal specimen
permanent
blue
nuclei of animal cells
24
Advantages of using differential stains
-most biological specimens are colourless and almost transparent so differential stains make it easier to observe tissues/cells/chemicals
-when observing plant tissue= allows observer to distinguish chemicals enabling the identification of different tissue types such as xylem vessels from phloem
-when observing animal tissue, it allows observer to distinguish between different types of white blood
cell
-improves contrast between structures
25
Advantages of LM
-low skill set needed by user
-can be transported to use in field work
-can observe living organisms
-relatively inexpensive so available for schools and colleges
26
Disadvantages of LM
-low resolution
-limited magnification
many internal cellular structures can't be seen such as ribosomes, cristae
27
Romanowsky Stains-Leishman's stain
Allow blood smear to AIR dry
Fix with methanol
Flood the slide with Leishman's stain
Leave for 2 mins
Dilute with distilled water
Leave for 7-10 minutes to AIR dry
Wash with DISTILLED water until the smear appears pale pink to the naked eye
28
Key points of cell theory
A cell is the basic unit of all life forms.
All living organisms are made up of one cell (unicelluar organisms) or more than one cell (multicelluar)
Metabolic processes occur inside living cells.
All new cells are derived from pre-existing cells.
Cells possess genetic material which can be passed on to their daughter cells.
A cell is the smallest unit of an organism capable of surviving independently.
29
What is the impact of microscopes on biology?
-enables scientists to see and examine cells in detail
-opened up new fields of science
30
Electron microscopes
Electron microscopes:
-use a beam of electrons which have shorter wavelength
-electron beam is focussed by electromagnetic condenser lens
-achieves greater resolution than LM which enables higher magnification which enables finer detail to be seen
-electrons focussed on fluorescent screen which emits visible light where electrons hit
-image converted to photomicrograph
-final images are always black, grey and white
31
Type of electron microscope: Transmission EM
TEM:
-produces images of cell ultrastructure and smaller organelles
-specimens must be very thin--->use ultra-microtome as electrons have to pass through the specimen
-specimens require complex staining process
-heavy metals used to stain specimen as atoms of heavy metals have large, positive nuclei that scatter electrons
-artefacts can occur due to the preparation techniques
-scattered electrons don't hit the fluorescent screen which means there is a dark area on image
cell structures appear as dark images
2D and black and white images
higher magnification=x500,00higher resolution=0.1nm
enables identification of organelles and observation of internal structures
provides detail of surface composition
32
Scanning Electron Microscope
-produces image of cell surface and topography
-beam of electrons is passed backwards and forwards over specimen in regular pattern
-electrons reflected off the surface of the specimen
pattern of scattered electrons reflects the contours of the specimen
no internal structures observed-only external detail
information processed by computer to generate 3D image
max magnification-100,000
lower resolution than TEM but higher than LM at 10nm
33
Confocal Laser Scanning Microscope
-can produce focused images of thick specimens at various depths due to optical sectioning
-point source of light is detected on to the object plane
point of emitted fluorescence light or reflected light from the specimen is directed through the director pinhole
fluorescent light is enhanced using photomultiplier
fluorescent light displayed on computer screen as a pixel
specimen is scanned point by point and line by line-very thin, blur-free optical sections are recorded one pixel at a time
-series of images is combined to form an image stack
34
How are fluorescent markers used to detect biological chemicals/structures?
-fluorochromes are attached to antibody that is specific for one antigen on or in the cell
-fluorochromes can also be tagged to a chemical that binds specifically to a component of the csm, DNA or other structure.
each fluorochrome has its own peak excitation and emission.
lasers with different wavelengths are used depending on which fluorochromes are used.
laser excites the fluorochrome causing the tagged cells to fluoresce.
this fluorescence is counted by the detector.
this specific light scattering and fluorescent characteristics of each cell as they pass the laser beam is used for counting and sorting.
35
Images formed by CLSM
Images are high resolution as very thin sections are examined and the light from elsewhere is removed
2D images as the illuminating spot is moved across the surface of the specimen
can be 3D when images are created at different focal planes.
36
Advantages of Electron Microscope
-greater resolution
-higher magnification
-finer ultrastructure and detail seen e.g ribosomes
37
Disadvantages of EM
-specimen has to be placed in a vacuum therefore must be dead
highly expesnive so prohibits use in schools
high skill set required as training is needed before use
must be used in specialist room due to electromagnets within the EM
more complex process to prepare specimen e.g must be mounted on copper grid rather than glass slide
more prone to artefacts due to complex preparation procedure
38
Advantages of CLSM
-Ability to reduce or eliminate background information away from the focal plane to reduce to image degradation
capability to collect a series of optical sections from thick specimens as various depths by optical sectioning
high resolution images
3D reconstructions
living cells can be used
any out-of-focus light is blocked for a clearer image than standard LM
39
How to use a LM?
-Insert x10 eyepiece lens into eyepiece into top of barrel
-Place slide on stage and secure in position using stage clips
-Select lowest power objective lens on the turret.
-Whilst looking at the stage and slide from the slide of the microscope: use coarse adjustment knob to bring stage up to the highest setting beneath the slide
-Look down the eyepiece: using the fine adjustment knob move the stage slowly downwards until specimen is roughly in focus.
-Adjust very slowly with the fine adjustment knob until the specimen is in clear focus.
If a greater magnification is required, ensure that the specimen is in the centre of the field of view.
Select higher objective lens ( medium power x10)
Refocus the specimen by adjusting the fine adjustment knob very slowly until the specimen is once again in clear focus.
Repeat at higher power (x4) if required.
40
Erythrocytes: typical abundance, size and cytoplasm contents
-approx 5 million per mm cubed
6.2-8.2 micrometre diameter
2-2.5 micrometre thick
-contains approx 250 million molecules of haemoglobin
-lack nucleus
-lack most other organelles
-contain carbonic anhydrase
-biconcave shape-flexibility to squeeze through capillaries
-large SA:vol
41
Erythrocytes
-Function
-Details of production
-transport oxygen as oxyhaemoglobin from lungs to tissues
-transport carbon dioxide from tissues to lungs
-produced in bone marrow by erythropoiesis which is stimulated by the hormone erythropoietin (EPQ)
-Immature rbc=reticulocytes
-Last approx 100-200 days
42
Thrombocytes:
-Typical abundance
-Size
-Cytoplasm contents
-approx 250000 per mm cubed
-2-3 micrometre diameter
-cytoplasmic fragments
-no nucleus
-irregular shape
- approx 1/4 size of rbc
-biconvex shape
43
Thrombocytes:
-Function
-Details of production
-role in blood clotting
-role in clot formation
-produced in bone marrow
-lasts approx 6-7 days
-destroyed in spleen and liver
44
Neutrophils:
-Typical abundance
-Size
-Cytoplasm contents
-approx 7000 per mm cubed
-12-17 diameter
-granulocyte
-high number of lysosomes
-multilobed (2-5 lobed) nucleus to increase flexibility
-can't renew their lysosomes
45
Neutrophils:
-Function
-Details of production
-defend against bacterial and fungal infections
-engulf bacteria by phagocytosis
-made in bone marrow
-squeeze out of the capillary fenestrations by amoeboid movement
-die after approx 5 days after carrying out phagocytosis
46
B-lymphocytes:
-Typical abundance
-Size
-Cytoplasm contents
-approx 7000 per mm cubed
-10-15 diameter
-agranulocyte
-large nucleus
-small quantity of cytoplasm
47
B-lymphocytes:
-Function
-Details of production
-produce immunoglobulins (antibodies)
-produce in the bone marrow
-B cells migrate to the spleen and other secondary lymphoid tissues where they mature and differentiate into immunocompetent B cells
48
T-lymphocytes:
-Typical abundance
-Size
-Cytoplasm contents
-approx 7000per mm cubed
-7-8 diameter
-agranulocyte
-large nucleus
-small quantity of cytoplasm
49
T-lymphocytes:
-Function
-Details of production
-several types
-T helper cell-produces cytokines to coordinate the immune response
-cytotoxic T cell-binds to antigens on viral infected cells/tumour cells and destroys them
-natural killer cells-roles vary
-T cells are produced in the bone marrow
-Later move to the thymus gland where they mature
50
Monocytes:
-Typical abundance
-Size
-Cytoplasm contents
-approx 7000per mm cubed
-10-30 micrometres in diameter
-agranulocyte
-kidney bean shaped nucleus
-largest of all leucocytes
-have the ability to replace their lysosomes
-nucleus:cytoplasm ratio ranges from 2:1 to 1:1
51
Monocytes:
-Function
-Details of production
-carry out phagocytosis
-longer living than neutrophils
-can leave the blood stream by amoeboid movement and differentiate into macrophages in the liver, spleen and lungs
-produced in the bone marrow
-spend approx 30-40h in blood before becoming macrophages
(macrophages develop from monocytes after they have left the bloodstream and exist in tissues whereas monocytes are in the circulation system)
52
What type of process is protein synthesis?
-anabolic process (builds smaller molecules up into larger molecules)
53
Compartmentalisation
-separation of specific areas inside the cell into self-contained units
54
What does enabling the cell to show division of labour do?
-increases productivity and efficiency
55
Microfilaments
-made from specialised protein (actin)
-thinnest cytoskeletal filaments
-consists of 2 intertwined strands
-7nm width
-maintains the cell shape
-enable motility e.g pseudopodia
-enable muscle contraction e.g skeletal muscle
-enable cytokinesis of cell divison
56
Intermediate filaments
-10nm diameter
-more stable than microfilaments
-made specialised protein called keratin
-consist of fibres wound together
-maintains the cell shape
-anchor the nucleus and organelles in the cytosol
57
Microtubules
-23nm diameter
-made of specialised protein called tubulin
-arranged in hollow cylinders
-arranged in 9+2 arrangement in cilia
-outer 9 are pairs of microtubules (or doublets)
-central 2 are single microtubules
-maintains the cell shape
-enables motility e.g cilia and flagella
-enables movement of chromosomes
-enables movement of organelles
58
Function of all 3 filaments
-help to maintain the cell's shape and structure
-enable the movement of organelles within the cytosol
enable the intracellular transport of molecules and materials
-enable the movement of chromosomes to occur during mitosis and meiosis
59
Motor proteins
-found in the cytoplasm
acts as molecular motor proteins, cross links and nucleation promoting factors
-e.g myosin (found in skeletal muscles): important in muscle contraction (sliding filament theory)
-others required to actively transport: proteins, membrane-bound organelles, vesicles
-Motor proteins can bind to cytoskeletal microtubules or actin filaments and move along them using energy released from the hydrolysis of ATP.
60
Interrelationship between organelles
Nucleolus: role in protein production
-synthesis of ribosomal subunits
-assembly of ribosomes
61
Nucleus: role in protein production
-contains chromosomes which contain genes coding for synthesis of polypeptide chains
-site of mRNA synthesis
62
Nuclear pore: role in protein production
-allows exit of mRNA after transcription
-allows exit of assembled ribosomes
63
RER: role in protein production
-site of polypeptide chain synthesis and hence protein synthesis
-proteins pass to cistern of ER to be packaged and transported to Golgi apparatus
64
Ribosomes: role in protein production
-site of polypeptide synthesis and hence protein synthesis
65
Mitochondria :role in protein production
site of aerobic respiration=production of ATP
hydrolysis of ATP releases energy to activate RNA nucleotides (to make rRNA) and activate tRNA molecules (to make polypeptide chains)
66
Golgi apparatus
-processes, modifies and packages proteins
-forms Golgi vesicles to transport proteins around cell
-form lysosomes
67
Golgi vesicle
-transports proteins and lipids around cell cytosol
68
Lysosome
-contains hydrolytic enzymes to break down cell debris, unwanted molecules and ageing or damaged cells
69
Golgi apparatus
-processes, modifies and packages proteins
-forms golgi vesicles to transport proteins around cell
-forms lysosomes
70
Golgi vesicle
-transports proteins and lipids around cell cytosol
71
Lysosome
-contains hydrolytic enzymes to break down cell debris, unwanted molecules, and ageing or damaged cells
72
What is flow cytometry a widely used method for?
-determines the different cell types in heterogenous cell populations
-analyse cell size and volume
-analyses the activity of molecules found in the cell surface membrane within the cell
-determine the purity of samples
73
Compare the resolutions of TEM and SEM and the images formed by them
TEM has a higher resolution of 2nm whereas SEM has a resolution of 50nm
TEM shows image of cell interior and shows ultrastructure
SEM gives 3D image and shows cell surface
74
Explain why it is important to use a differential stain when examining a blood smear
-to identify differences between cells
-to identify differences between organelles
-red blood cells visible anyway without stain due to haemoglobin
-allows white blood cells to be counted
75
Describe how the rough endoplasmic reticulum and the Golgi apparatus are involved in the production of a secretory vesicle that contains protein
-proteins are synthesised on the ribosomes of RER
-proteins then pass into the lumen of RER
-proteins can have carbohydrates added
-proteins are packaged into transport vesicles
-transport vesicles move to Golgi by microtubules
-vesicles fuse with cis face of Golgi
-proteins are modified in Golgi and packaged into secretory vesicles
76
Outline the importance of cytoskeleton
-provides mechanical strength to the cell
-holds organelles in place
-aids transport of organelles such as cilia and flagellum
-cell movement
-maintains cell shape
-role in cell division
77
Fluid mosaic model
fluid=phospholipids and protein molecules can move about laterally within their monolayer
mosaic=protein molecules are interspersed and scattered within each monolayer
78
Where do cell membranes exist?
-around the outside of the cell
-within the cell e.g surrounding all membrane bound organelles in eukaryotic cells
79
Function of cell membranes
-control entry and exit of molecules within organelles
-isolate organelles to enable specialisation of chemical reactions
-provide internal transport system
-concentrate enzymes and substrates to increase efficiency and productivity of an organelle
-isolate enzymes to prevent cellular damage
-maintain internal specific conditions at optimal level
-can be moved within cell to where they are needed
-provide surfaces for chemical reactions
mitochondria, chloroplasts and nucleus can be found as double membranes forming an envelope
80
Properties of phospholipid bilayers
-consist of 2 monolayers with a combined width of 7nm wide
-hydrophobic core allows lipid soluble molecules to cross bilayer
-hydrophilic phosphate head prevents water soluble molecules from crossing bilayer
-allows membrane to be flexible
-allows membrane to be stable
81
Function of integral (intrinsic) proteins
Act as:
-carrier proteins for specific molecules
-channel proteins for water soluble molecules and ions
-electron carriers
-structural role
-receptor proteins
-membrane bound organelles
82
Function of peripheral (extrinsic) proteins
Act as:
-glycoproteins receptors
-membrane bound organelles
-electron carriers
-structural role
-cell to cell recognition
-antigens that trigger an immune response
-cell to cell adhesion
83
Phospholipid
-has a polar head which consists of glycerol, choline and phosphate group and is hydrophilic so forms hydrogen bonds with water which stabilises the membrane
-non polar tail which consists of 2 fatty acid tails and is hydrophobic (the more unsaturated fatty acids present, the more fluid the membrane as the unsaturated fatty acids prevent the close packing of the phospholipids at low temperatures which keeps cell membranes fluid which maintains movement of membrane proteins and maintains movement of substances across membranes)
-PPL molecules are arranged as a bilayer
-PPL bilayer acts as a barrier to water soluble molecules
84
Glycocalyx
-carbohydrate chain which can be added to a protein or a PPL
-acts as antigens
-acts as recognition sites for the attachment of other molecules
85
Glycolipid=lipid + glycocalyx
-acts as recognition sites (antigens and cell markers) for cell adhesion, interacts with water to stabilise the cell membrane
-e.g cholera toxin
86
Glycoprotein=protein+ glycocalyx
-acts as recognition sites for molecules (antigen s and cell markers) for cell adhesion
-e.g hormones and neurotransmitters
interacts with water to stabilise the membrane
-acts as a receptor for signalling molecule (cell recognition)
87
Non-transport protein
-cell to cell adhesion
-enzyme or co enzyme
-anchoring the cytoskeleton
88
Transport protein
Carrier protein: for active transport or facilitated diffusion
carrier or channel protein allows facilitated diffusion
89
Cholesterol
-stabilises the membrane
-regulates the fluidity of the membrane (at low temp cholesterol increases fluidity as it prevents close packing of the PPLs hence membrane is more flexible, at high temps cholesterol decreases fluidity and so stabilises the membrane as membrane is less flexible)
-prevents passage of ions and polar/hydrophilic molecules through the membrane
90
Passive movement across cell membranes: simple diffusion
-used to transport molecules which are small and lipid soluble
-e.g oxygen, ammonia, alcohol, CO2, fat soluble vitamins, CO
-rate of simple diffusion depends on: concentration gradient, size of molecule, kinetic energy the molecule possesses, polarity of molecule, surface area)
91
Passive movement across cell membranes: facilitated diffusion
a) channel proteins: uses intrinsic proteins which form channels to provide hydrophilic pathways for ions or pores
-pores form aqueous channels which allows ions to cross which can be gated which enables the channels to be open or closed depending on the p.d across membrane and the presence of a specific molecule (ligand)
-used to transport water soluble molecules and ions
-different channels are specific to specific ions
rate of facilitated diffusion affected by same factors as simple diffusion but also the number of channel proteins in cell membranes
92
Facilitated diffusion: carrier proteins
-uses intrinsic proteins which form specific carriers
-each carrier protein has a complementary binding site to a specific molecule
-carrier protein undergoes allostery (a conformational change)
-carrier protein changes between a ping and pong state
-ping state= binding site exposed to inside of cell
-pong state=binding site exposed to outside of cell
-used to transport larger molecules
-rate of facilitated diffusion affected by same factors as simple diffusion but also the number of channel proteins in cell membrane
93
Active movement across cell membranes: active transport
-transports molecules against a concentration gradient
-requires an additional input of ATP as the kinetic energy of the molecules is insufficient
-energy obtained by hydrolysis of ATP produced in aerobic respiration
-requires the use of intrinsic carrier proteins within the phospholipid bilayer
-each carrier protein is specific for one or a few molecules
-carrier protein also has a separate binding site for an ATP molecule
energy required to change shape of the carrier protein is allostery
cells can carry out active transport the cell must have a high number of mitochondria to produce ATP
94
Active transport
-molecules to be transported inside cell (carrier protein in lipid bilayer)
- carrier protein activated by reaction with ATP
-change in shape and position of carrier protein so molecule is released releasing ADP and Pi
-ADP and Pi released from carrier protein which reverts to the receptive shape
95
How are carrier proteins involved in facilitated diffusion and active transport similar?
-both are intrinsic proteins
-possess a binding site
-have a binding site which is specific for a particular molecule
-have a binding site which is complementary to the molecule to be transportyed
-undergo a conformational change (allostery) to move the molecules
96
How are carrier proteins involved in facilitated diffusion and active transport different?
-additional binding site for ATP
-requires the hydrolysis of ATP to release the molecule form the binding site
-only work in one direction
-allow the accumulation of molecules within the cell
-work against the concentration gradient
97
Endocytosis
a) Phagocytosis: when cells (phagocytes) take in solid material in large quantities and produce phagocytic vesicles
e.g bacteria being engulfed by a phagocyte
b) Pinocytosis: when cells taken to liquid material in large quantities and very small vesicles are formed in a process called micropinocytosis e.g the uptake of nutrients by an ovum from follicle cells
98
Exocytosis
the removal of waste products or secretion of useful products)e.g secretion of pancreatic enzymes, secretin of insulin from beta cells of the pancreas
-plant cells use it to develop the cell wall which is outside of the cell surface membrane
99
Qualitative test
-only tells of presence or absence of substance
-monosaccharides and disaccharides dissolve in water so will not form a precipitate
100
Semi-quantitative test
-the quantity of each sample should be controlled
-resulting positive result should be scaled low/ medium/high
height of emulsion formed could be used to give an indication of the quantity of lipid
101
Quantitative emulsion test
-method should be adapted to measure the density of the emulsion in a well-mixed sample using a colorimeter
102
Colorimeter practical
-Using a syringe add 10cm cubed of distilled water to 5 labelled test tubes each
-acclimatise one test tube in each of the 5 thermostatically controlled water baths for 5 minutes to allow the water to equilibrate to the correct temperature
-collect 10 beetroot cylinders and trim them all to 30mm
-rinse cylinders under a running tap
-add 2 cylinders to each tube in each temperature and leave for 15 minutes
-label the cuvettes with temps from 20-60 degrees
-remove the tubes from the water bath and pour the remaining liquid into a cuvette
-use the colorimeter to measure the absorption for each temperature]-plot a graph of temperature against absorption and draw an appropriate line
103
Describe and explain the results shown by this practical
-high temperatures (e.g 55 degrees) increased the permeability of the tonoplast and plasma membrane
-membrane proteins become denatured
-both these create spaces for pigment leakage by diffusion
-occurs faster as more pigment leaks out at high temperatures
-non linear trend
104
How do solvents affect membrane permeability?
-ethanol will cause membrane disintegration as it forms temporary bonds with the phospholipid heads in the membranes
-causes the phospholipids to move out of place so large gaps form in the membrane allowing pigment to leak out
-ethanol also affects bonding in the membrane proteins leading to denaturation causing further gaps in the membrane
-cholesterol important in membrane structure as it is soluble in ethanol and so once it is removed, large gaps allow increased permeability of molecules such as betanin
105
Limitations of beetroot practical
-cutting cylinders will damage some cell walls and tonoplasts which will vary between different cylinders
-differences in blotting techniques (more blotting=removes more pigment)
-cylinders sit on top of each other which decreases the surface area exposed to water
106
Errors of beetroot practical
-accuracy of cutting cylinders= decreases in surface area
-accuracy of timing period
107
Improvements for beetroot practical
-repeat 3 times
-ensure all cylinders cut from same area of the same beetroot
-standardise blotting mechanism e.g place on towel and roll once
-stir coloured liquid after removing cylinder before decanting
-decant coloured liquid through sieve into cuvette to remove any tissue debris
108
Principles of colorimetry investigation: temperature
-As the temperature of the cells increases, the kinetic energy of the phospholipid molecules increases causing the PPL molecules to move more within their monolayer, causes them to be more spaced out, causes spaces to occur between the PPL molecules within the cell membranes which causes the cell surface membrane and the tonoplast to become disrupted
-the kinetic energy of the protein molecules increases, causing the protein molecules to move more within their monolayer, causes the protein molecules to become denatured, causes space to occur between the protein and PPL molecules within the cell membranes which causes the cell surface membrane and the tonoplast to become more disrupted
109
What happens when the kinetic energy of the betannin molecules increases?
-the betanin molecules move faster
-disruption of the tonoplast means the more energetic betanin molecules are released from the large central vacuole inside the cytosol
-disruption of the cell surface membranes means the more energetic betanin molecules are released from the cytosol int the surrounding external solution
-combined effect of all of the above is that the betanin molecules diffuse out of the vacuole and the cell both at a faster rate and in greater quantities
-solution becomes more densely coloured
110
Outline the roles of membranes at the surface of cells and within cells
At surface membranes:
-separates cell from environment
-controls entry and exit of molecules
-use of phospholipid layer
-facilitated diffusion
-active uptake
-phagocytosis, endocytosis
-cell recognition
-cell to cell attachment
-receptor for hormones and neurotransmitters
-microvilli increase surface area of cell
Within cells: compartmentalisation
-prevents disruption of reactions
-reactions take place on membrane
-enzymes attached to membranes
-isolates DNA
-nuclear pore permits RNA to leave nucleus
--forms ER
-attachment of ribosomes
-intracellular transport
-protects cells from contents of lysosomes
-tonoplast surrounds
-increases surface area of organelle
111
