Studying cells Flashcards
Methods of studying cells
Microscopy
Cell fractionation
What is cell fractionation?
A method of studying cell organelles by separating them from the cell
Step 1 of cell fractionation
Take a sample of cells and HOMOGENISE it in a blender with a buffer solution to break up cells (NOT ORGANELLES) and release organelles that are being studied
Properties of buffer solution
Cold
pH constant
Water potential in solution same as inside the cell (isotonic)
Why does the buffer solution have to be cold?
To keep enzyme activity low so destructive enzymes prevented from engulfing
Why does the buffer solution have to have a constant pH?
Because if pH changed, then the enzymes in organelles would denature and damage organelles
Why does the buffer solution have to have same water potential as inside cell
Prevents osmosis of water into and out of organelles causing them to swell or shrink respectively = damage
What is left after homogenisation?
The homogenate. Contains unbroken cells and different density organelles
What is done with the homogenate?
Put in a centrifuge to spin at relatively high speed
Balance out with same volume of sample in other tube
What is left after spinning at relatively high speed?
The larger organelles which experienced more force sunk to bottom (pellet)
But mid-small organelles remain in solution, not spun fast enough to sink (supernatant)
What is done with the supernatant and pellet?
Pour out supernatant so first tube is left with pellet of separated high density organelle
Why is the supernatant spun at increasingly high speeds?
So smaller density organelles are separated from solution and form a pellet to be separated
What should pellets be kept at?
Ice cold temperature to reduce enzyme activity
Will all organelles be separated perfectly?
No because pellets always contain traces of others
High density organelles
Nucleus
Golgi
Endoplasmic reticulum
Cell surface membranes
High- mid density organelles
Mitochondria
Mid- low density organelles
Lysosomes
Low density organelles
Ribosomes
Light microscopes
Using visible light to pass through specimens to view layers of cells with organelles
Eyepiece lens
View specimen here w eye piece graticule
Coarse focus
Moves stage up and down to focus it
Objective lens
Changes magnification by switching lens
Fine focus
Fine tune focus without moving the stage
Resolving power
Ability to distinguish between 2 separate objectives
Minimum distance between 2 objects where they are still seen as 2 separate objects
Magnification on a scale bar
Measure length of scale bar
Find what actual size it represents
Mag = image size/actual size
Actual size of an image
Find conversion of length of scale bar to actual size it represents
Measure image size of image
Use conversion to find its actual size
Magnification =
Image size/actual size
Why do we need to calibrate the stage micrometer?
Because we are unsure of the the scale on stage micrometer so can not be used to measure organelles/cells
What item do you use to calibrate an eyepiece graticule?
A stage micrometer
How to calibrate eyepiece graticule with stage micrometer
View stage micrometer under same magnification you will measure cell in
Ensure division of stage micrometer is known, eg each small division =1um
Find point where divisions line up to make conversion
Measure organelle with eyepiece graticule and use this scale
What type of image does light microscope produce?
2D image
Able to view natural colour
Magnification of a light microscope calculate by?
The eyepiece lens (always same in one microscope) x objective lens chosen
Magnification of light microscope
Max of 1000x
Is light microscope able to view living specimens?
Yes
Resolution of light microscope
Low = around 200nm
So if objects less than 200nm they are not distinguished as 2 separate objects
2 types of electron microscope
Transmission (TEM)
Scanning (SEM)
How do electron microscopes work?
Using electron beams instead of light
Transmission electron microscope works by?
Passing electron beams through a specimen then producing image on a screen
Image produced by TEM
2D image
In black and white
Can TEMs view living specimens?
No, because beams pass through species in a vacuum
What must specimens be in TEMs?
Thinly sliced
Dead
How do SEMs work?
Electrons don’t pass through specimen yet scattered on the surface of the specimen to produce an image once detected
Image produced by SEMs
Produces 3D image
In black and white
Magnification of TEMs
Very very high
High resolving power
What is a temporary mount?
Placing a specimen on a slide with a drop of water then coverslip on top
Held by surface tension
How are temporary mounts made?
Place a drop of water on a glass slide
Use forceps to place a THIN LAYER or specimen on it
Lower coverslip at an angle to reduce air bubbles
When using a light microscope, how do you find the mean number of certain organelles in a cell?
Select a large number of cells at random
Count the number of organelles in each cell
Divide this total by number of cells selected
Why do light microscopes have lower resolving power than electron microscopes?
Because light has a longer wavelength than electron beams
Advantages of light microscope
Can see living specimens
Easier to prepare specimens
Variety of coloured stains
Disadvantages of light microscope
Low resolution so less detailed and unable to view smaller components
Advantages of TEM
Very high resolution at a higher magnification
Detail of organelles
Disadvantages of TEM
dead specimens in a vacuum
difficult to prepare by creating very thin specimens
produces black and white image
artefacts spoil the image possibly
Advantages of SEM
3D images can show structural formation of cells
Disadvantages of SEM
Specimens dead
Hard to prepare
Black and white image
