Studying cells

Question 1

Methods of studying cells

Answer 1

Microscopy
Cell fractionation

Question 2

What is cell fractionation?

Answer 2

A method of studying cell organelles by separating them from the cell

Question 3

Step 1 of cell fractionation

Answer 3

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

Question 4

Properties of extraction solution

Answer 4

Cold
pH constant
Water potential in solution same as inside the cell (isotonic)

Question 5

Why does the buffer solution have to be cold?

Answer 5

To keep enzyme activity low so destructive enzymes prevented from digesting organelles

Question 6

Why does the extraction solution have to have a buffered pH?

Answer 6

Because if pH changed, then the enzymes in organelles would denature and damage organelles

Question 7

Why does the extraction solution have to have same water potential as inside cell

Answer 7

Prevents osmosis of water into and out of organelles causing them to swell or shrink respectively = damage

Question 8

What is left after homogenisation?

Answer 8

The homogenate. Contains unbroken cells and different density organelles

Question 9

What is done with the homogenate?

Answer 9

Put in a centrifuge to spin at relatively high speed
Balance out with same volume of sample in other tube

Question 10

What is left after spinning at relatively high speed?

Answer 10

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)

Question 11

What is done with the supernatant and pellet?

Answer 11

Pour out supernatant so first tube is left with pellet of separated high density organelle

Question 12

Why is the supernatant spun at increasingly high speeds?

Answer 12

So smaller density organelles are separated from solution and form a pellet to be separated

Question 13

What should pellets be kept at?

Answer 13

Ice cold temperature to reduce enzyme activity

Question 14

Will all organelles be separated perfectly?

Answer 14

No because pellets always contain traces of others

Question 15

High density organelles

Answer 15

Nucleus
Golgi
Endoplasmic reticulum
Cell surface membranes

Question 16

High- mid density organelles

Answer 16

Mitochondria

Question 17

Mid- low density organelles

Answer 17

Lysosomes

Question 18

Low density organelles

Answer 18

Ribosomes

Question 19

Light microscopes

Answer 19

Using visible light to pass through specimens to view layers of cells with organelles

Question 20

Eyepiece lens

Answer 20

View specimen here w eye piece graticule

Question 21

Coarse focus

Answer 21

Moves stage up and down to focus it

Question 22

Objective lens

Answer 22

Changes magnification by switching lens

Question 23

Fine focus

Answer 23

Fine tune focus without moving the stage

Question 24

Resolving power

Answer 24

Ability to distinguish between 2 separate objectives
Minimum distance between 2 objects where they are still seen as 2 separate objects

Question 25

Magnification on a scale bar

Answer 25

Measure length of scale bar Find what actual size it represents Mag = image size/actual size

Question 26

Actual size of an image

Answer 26

Find conversion of length of scale bar to actual size it represents Measure image size of image Use conversion to find its actual size

Question 27

Magnification =

Answer 27

Image size/actual size

Question 28

Why do we need to calibrate the stage micrometer?

Answer 28

Because we are unsure of the the scale on stage micrometer so can not be used to measure organelles/cells

Question 29

What item do you use to calibrate an eyepiece graticule?

Answer 29

A stage micrometer

Question 30

How to calibrate eyepiece graticule with stage micrometer

Answer 30

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

Question 31

What type of image does light microscope produce?

Answer 31

2D image Able to view natural colour

Question 32

Magnification of a light microscope calculate by?

Answer 32

The eyepiece lens (always same in one microscope) x objective lens chosen

Question 33

Magnification of light microscope

Answer 33

Max of 1000x

Question 34

Is light microscope able to view living specimens?

Answer 34

Yes

Question 35

Resolution of light microscope

Answer 35

Low = around 200nm So if objects less than 200nm they are not distinguished as 2 separate objects

Question 36

2 types of electron microscope

Answer 36

Transmission (TEM) Scanning (SEM)

Question 37

How do electron microscopes work?

Answer 37

Using electron beams instead of light

Question 38

Transmission electron microscope works by?

Answer 38

Passing electron beams through a specimen then producing image on a screen

Question 39

Image produced by TEM

Answer 39

2D image In black and white

Question 40

Can TEMs view living specimens?

Answer 40

No, because beams pass through species in a vacuum

Question 41

What must specimens be in TEMs?

Answer 41

Thinly sliced Dead

Question 42

How do SEMs work?

Answer 42

Electrons don’t pass through specimen yet scattered on the surface of the specimen to produce an image once detected

Question 43

Image produced by SEMs

Answer 43

Produces 3D image In black and white

Question 44

Magnification of TEMs

Answer 44

Very very high High resolving power

Question 45

What is a temporary mount?

Answer 45

Placing a specimen on a slide with a drop of water then coverslip on top Held by surface tension

Question 46

How are temporary mounts made?

Answer 46

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 using a mounted needle

Question 47

When using a light microscope, how do you find the mean number of certain organelles in a cell?

Answer 47

Select a large number of cells at random Count the number of organelles in each cell Divide this total by number of cells selected

Question 48

Why do light microscopes have lower resolving power than electron microscopes?

Answer 48

Because light has a longer wavelength than electron beams

Question 49

Advantages of light microscope

Answer 49

Can see living specimens Easier to prepare specimens Variety of coloured stains

Question 50

Disadvantages of light microscope

Answer 50

Low resolution so less detailed and unable to view smaller components

Question 51

Advantages of TEM

Answer 51

Very high resolution at a higher magnification Detail of organelles

Question 52

Disadvantages of TEM

Answer 52

dead specimens in a vacuum difficult to prepare by creating very thin specimens: some organelles may be missed simply because they were not present in the slice taken produces black and white image artefacts spoil the image possibly

Question 53

Advantages of SEM

Answer 53

3D images can show structural formation of cells

Question 54

Disadvantages of SEM

Answer 54

Specimens dead Hard to prepare Black and white image

Question 55

Stain used to view starch granules

Answer 55

Iodine in Potassium iodide

Question 56

How to tell if an optical microscope vs TEM was used?

Answer 56

transmission electron microscope has higher resolution so allows for internal structure of organelles to be seen

Question 57

What is an artefact?

Answer 57

Structure observed in a cell using a microscope or experiment that is not naturally present but occurs as a result of damage to specimen when being prepared

Question 58

What stops electron microscopes achieving maximum resolution?

Answer 58

Changes to the specimen during the preparation process can limit the resolution Higher energy electron beams give even shorter wavelength and therefore even greater resolution, but these can also damage the specimen