Topic 2 cells- methods of studying cells Flashcards
During homogenisation, why must the cells be in an ice cold, isotonic and buffered solution?
Ice-cold: to reduce enzyme activity. When cell is broken open, enzymes are released which could damage the organelles.
Isotonic: organelles must be the same water potential as the solution to prevent osmosis, as this could cause the organelles to either shrivel or burst.
Buffered: The solution has a buffer pH to prevent organelle proteins from becoming denatured.
What are the stages of cell fractionation?
- homogenisation
- filtration
- ultracentrifugation
What is the purpose of cell fractionation?
Cell fractionation is used to isolate different organelles so they can be studied. This enables individual organelle structures and functions to be studied.
Describe the stages of cell fractionation and ultracentrifugation
- The cells are homogenised to break open cells, in an ice-cold, isotonic and buffered solution. Solution ice-cold to reduce damage by enzymes. Solution isotonic to prevent osmosis, as this could cause organelles to either shrivel or burst (prevent damage to organelles). Solution buffered to prevent protein denaturation.
- filter to remove large debris
- spin filtrate in centrifuge at low speed and the densest organelles will form pellet at bottom
- Repeat process, by spinning supernatant at increasingly higher speeds, until all organelles are separated.
Describe and explain how cell fractionation and ultracentrifugation can be used to isolate mitochondria from a suspension of animal cells.
- cell homogenisation to break open cells
- use isotonic solution to prevent damage to mitochondria
- keep solution cold to prevent prevent damage by enzymes
- keep solution buffered to prevent organelle protein denaturation
- centrifuge at low spped to separate nuclei/heavier organelles
- respin supernatant after pellet removed at a higher speed to get mitochondria to from pellet at bottom.
What are the ADVANTAGES of TEMs?
High resolution images, which allows internal structures within cells (or even within organelles) to be seen.
What are the disadvantages of TEMs?
-They can only be used on very thin specimens
- They cannot be used to observe live specimens
- Lengthy treatment required to prepare specimens means that artefacts can be introduced (look like real structures but are actually the results of preserving and staining)
- do not produce colour images, unlike optical microscopes
What are the ADVANTAGES of SEMs?
-Can be used on thick/ 3D specimens
- Allow the external 3D structure of specimens to be observed
What are the DISADVANTAGES of SEMs?
-Lower resolution images that TEMs
- cannot observe live specimens, unlike optical microscopes
- do not produce coloured images, unlike optical microscopes
How do you work out the magnification/Actual size/Image size?
Magnification= image size/actual size
Actual size= image size/magnification
Image size= actual size X magnification
How do you Calibrate the Eyepiece graticule?
- Line up the stage micrometer and eyepiece graticule, whilst looking through the eyepiece
- Count how many divisions on eyepiece graticule fit into one division on micrometer scale.
- Each division on the micrometer is 10 micrometres, so this can be used to calculate what one division on the eyepiece graticule is at that current magnification.
- once graticule calculated, you can measure the size of the cells/organelles.
What is Resolution?
The minimum distant between 2 objects, in which they can still be viewed as separate
- Resolution in optical microscopes is determined by the wavelength of light
-Resolution in electron microscopes is determined by the wavelength of the beam of electrons.
Describe the differences between Optical (light) microscopes and Electron microscopes
-Optical microscopes use light to form an image, whereas Electron microscopes use electrons to form an image
- The maximum resolution of optical microscopes is around 200 nm, whereas the maximum resolution of of electron microscopes is around 1000 times more.
- The use of electrons in electron microscopes means there is an increased resolution, as electrons have a short wavelength, meaning there is a higher resolving power. There is limited resolution of optical microscopes, as it’s impossible to distinguish between two objects closer than half the wavelength of light (500-650 nm)
- Optical microscopes cannot be used to observe smaller organelles (endoplasmic reticulum, ribosomes, lysosomes), but electron microscopes can.
- The maximum magnification of optical microscopes is about x1500, but the maximum magnification of electron microscopes is about x1,500,000
- optical microscopes produce colour images, whereas electron microscopes produce black and white images
- Optical microscopes can be used to observe living specimens, whereas electron microscopes can only be used to observe non-living specimens, as the sample must be in a vacuum.
What is magnification?
How many times larger the image produced by the microscope is, compared to the object.
TEMs (Transmission electron microscopes)
-Extremely thin specimens are stained and placed in a vacuum.
- Electron gun produces beam of electrons that pass through specimen
- some parts absorb the electrons and appear dark
- Image produced is two-dimensional and shows detailed images on the internal structures of cells
SEMs (scanning electron microscopes)
-Specimens do not need to be thin, as the electrons are not transmitting through
-Instead, the electrons are beamed onto the surface and the electrons are scattered in different ways depending on the contours.
- This produces a three-dimensional image.
