microscopy / mitosis Flashcards
Describe how optical microscopes work.
- lenses focus rays of light + magnify the view of a thin slice of specimen.
- different structures absorb different amounts + wavelengths of light.
- reflected light is transmitted to the observer via the objective lens + eyepiece.
Suggest the advantages and limitations of using an optical microscope.
- colour image ,
- visualise living cells - watch behaviours like cell division in real time.
- affordable apparatus
- 2D image
- lower resolution than electron microscopes = cannot see ultrastructure .
- resolution 0.2μm - not large enough to visualise any smaller organelles
- magnify 1500x actual size
Describe how a transmission electron microscope (TEM) works.
1) uses electromagnets - pass high energy beam of electrons through thin slice of specimen.
(e- have shorter wavelength compared to visible light so higher resolution/ detailed images produced.)
2) more dense structures appear darker since they absorb more electrons.
3) focus image onto fluorescent screen / photographic plate using magnetic lenses .
Suggest the advantages + limitations of using a TEM.
- electrons have shorter wavelength than light = high resolution, so ultrastructure visible
- high magnification (x500000)
- high resolution (0.0002 μm/ 20nm)
but
- 2D image
- requires a vaccum - cannot show living structures extensive preparation may introduce artefacts // live cells cannot be used
- no colour image
Describe how a Scanning Electron Microscope (SEM) works.
1) emit a beam of electrons towards sample,, (knocking electrons off it used to build image ) onto a specimens surface electromagnetic lenses.
2) reflected electrons hit a collecting device and are amplified to produce an image on a photographic plate.
Suggest the advantages and disadvantages of using an SEM.
- produce 3D images of cells + organelles.
- electrons have shorter wavelength than light = high resolution.
but
- requires a vaccum = cannot show living structures
- no colour image
- only shows outer surface
- expensive
Define Magnification.
- how enlarged the image is compared to the original object
Define the term Resolution.
- how well a microscope distinguishes between two points that are close together.
Explain how to use an eyepiece graticule and stage micrometer to measure size of a structure.
1) place micrometer on stage to calibrate eyepiece graticule.
2) line up scales on graticule and micrometer. count how many graticule divisions are in 100um on the micrometer
3) length of 1 eyepiece division = 100um/ no. divisions
4) use calibrated values to calculate actual length of structures.
state an equation to calculate actual size of a structure from microscopy .
actual size = image size / magnification.
explain how to view specimens under an optical microscope .
1) pipette drop water onto slide then place specimen on top.
2) add drop of stain = creates contrast + enables organelles to be visualised .
3) add cover slip to protect specimen - carefully tilting + lowering down (careful no air bubbles)
4) place slide onto stage + select lowest-powered objective lens.
5) look down at eyepiece + use coarse adjustment knob to focus specimen
6) select increasingly higher magnification until visualise cell structures .
explain what is meant by cell fractionation.
- technique - separates organelles according to their density - to visualise certain organelles under microscope separately
- bursting cell surface membrane to release organelles + spinning cell solution at high speeds
outline what happens during cell fractionation and ultracentrifugation
1) mince and homogenise tissue to break open cells + releases organelles
2) filter homogenates to remove debris
3) perform differential centrifugation
a) spin homogenate in centrifuge
b) the most dense organelles on mixture will form a pellet
c) filter off the supernatant and spin again at higher speed
explain the first process of fractionation - homogenisation .
- break apart the plasma membrane to release the organelles
- vibrating the cells or by breaking them apart in a blender
—> needs placed in solution : ice-cold, isotonic and buffered .
why does solution need to be ice cold ?
- to slow down activity of enzymes
- some enzymes degrade organelles so need to reduce their activity to persevere cells organelles .
why solution need to be isotonic ?
- solute conc of solution need to be same as the cells that’s been broken down —> otherwise water would move into organelles by osmosis resulting in damage
why solution need to be buffered ?
- ensures pH stays constant .
- proteins are denatured by changes in pH
why does solution need to be buffered ?
- ensures the pH remains constant .
- proteins are denatured by changes of pH
why is the homogenised solution filtered ?
- to remove any tissue debris .
- organelles are small enough to pass through the holes of the filter paper so will be present in filtrate .
explain the process of ultracentrifugation .
- spin the filtrate at increasing speeds .
- heaviest organelles will sink to bottom of test tube forming a pellet
- transfer the remaining solution to a separate test tube (spun at slightly higher speed .)
- repeated until u obtain the organelle that u want = organelles separated from solution from heaviest to lightest .
define mitosis .
- type of cell division where cells produce identical copies of themselves + used for growth + repair and asexual reproduction .
state what the cel cycle is and outline its stages .
- cycle of division with intermediate growth periods
1) interphase
2) mitosis or meiosis (nuclear division )
3) cytokinesis ( cytoplasmic division )
explain why the cell cycle doesn’t occur in some cells .
- after differentiation , some types of cell in multicellular organisms no longer have ability to divide.
what’s the difference between the cell cycle and mitosis ?
- cell cycle includes growth period between divisions = mitosis is only 10% of the cycle + refers only to nuclear division .
outline what happens during interphase .
- individual chromosomes not visible - 2 chromatids contain 2 identical DNA molecules produced by semi conservative replication .
- G1: cell synthesis proteins for replication : e.g = tubulin for spindle fibres + cell size doubles
- S: cell replicates its DNA = chromosomes consists of 2 sister chromatids joined at a centromere .
- G2: cell keeps growing until all organelles have duplicated.
state the purpose of mitosis .
- produces 2 genetically identical daughter cells for :
- growth
- cell replacement / tissue repair
- asexual reproduction
outline what happens during prophase .
1) chromosomes condense : becoming visible . (X-shaped : 2 sister chromatids joined at centromere)
2) centrioles move to opposite poles of cell + mitotic spindle fibres form .
3) nuclear envelope + nucleolus disintegrates = chromosomes free in cytoplasm .
outline what happens during metaphase .
- sister chromatids line up along the middle of the cell .
- they attach to the spindle fibres by the centromere .
outline what happens during anaphase .
- the centromere splits + chromatids pulled to opposite poles of the cell .
- requires energy from ATP hydrolysis .
1) spindle fibres contract - centromeres divides
2) sister chromatids seperate into 2 distinct chromosomes + pulled to opposite poles of the cell
3) spindle fibres break down
outline what happens during telophase .
1) chromosomes decondense - becomes invisible again
2) new nuclear envelopes reforms around them forming 2 new nuclei , each with 1 copy of each chromosome
outline what happens during cytokinesis .
- cytoplasm divides = plasma membrane pinches off to form 2 new genetically identical cells .
describe how an uncontrolled mitosis may result to cancer .
- mitosis is genetically controlled + stops once cell divided enough times to make cells you need
- if genes that control mitosis mutate , mitosis can occur unchecked - resulting in formation of of a tumour .
—> if invades surrounding tissue - cause cancer .
how do some cancer cells work by disrupting the cell cycle ?
- some prevent synthesis of enzymes involved in DNA replication
—> prevents cell cycle progressing past S phase - undergo apoptosis - radiotherapy works by damaging DNA using radiation
- cell cycle stalled during DNA checkpoint stages
why is only the root tip used when calculating a mitotic index ?
- meristematic cells at root tip are actively undergoing mitosis.
- cells further from root tip are elongating rather than dividing .
how to calculate mitotic index ?
- measure of proportion of cells which are undergoing mitosis
- mitotic index = no. cells with visible chromosomes / total number of cells
suggest how cancer treatments control the rate of cell division .
- during cell cycle :
— prevent DNA relication .
— disrupt spindle information - inhibit metaphase / anaphase.
how do prokaryotes cells replicate ?
binary fission:
1) DNA loop replicates . Both copies stay attached to cell membrane . Plasmids replicate in cytoplasm .
2) cell elongates , separating the 2 DNA loops.
3) cell membrane contracts + septum forms
4) cell splits into 2 identical progeny cells: each with 1 copy of DNA loop but a variable no. plasmids.
estimate the exponential growth of bacteria within 8 hours. assume binary fission occurs once every 20 mins + there’s 1 bacterium at the start.
8 x 60 = 480 mins
480 / 20 = 24 divisions
2^24
why are viruses classified as non-living ?
- they are acellular - no cytoplasm, no metabolism + cannot self replicate.
outline how viruses replicate .
1) attachment proteins attach to receptors on host cell membrane .
2) enveloped viruses fuse with cell membrane or move in via endocytosis + release DNA/RNA into cytoplasm
3) host cell uses viral genetic information to synthesise new viral proteins / nucleic acid .
4) components of new viral particle assemble.
how do new viral particles leave the host cell?
- bud off + use cell membrane to form envelope .
- cause lysis of host cell.
why is it difficult to develop effective treatment against viruses ?
- replicate inside living cells = difficult to kill them without killing host cells
