3.2.1.3

Question 1

Outline how a student could prepare a temporary mount of tissue for an optical microscope

Answer 1

1. Obtain thin section of tissue
2. Place tissue in a drop of water
3. Stain tissue on a slide to make structures visible
4. Add coverslip using mounted needle at 45 degrees to avoid trapping airbubbles

Question 2

Suggest the advantages and limitations of using an optical microscope

Answer 2

+ colour image
+ can show living structures
+ affordable apparatus
- 2D image
- lower resolution than electron microscopes so cannot see ultrastucture

Question 3

Suggest the advantages and limitations of using a TEM

Answer 3

+ electrons have shorter wavelength than light = high resolution so ultrastructure visible
+ high magnification (x500,000)
- 2D image
- requires a vacuum = cant show living structures
-extensive preparation may introduce artefacts
-no colour image

Question 4

Suggest the advantages and limitation of using a SEM

Answer 4

+3D image
+ electrons have shorter wavelength than light = high resolution
-requires a vacuum = cannot show living structures
-no colour image
-only shows outer surface

Question 5

Define magnification and resolution

Answer 5

Magnification= the factor by which the image is larger than the actual specimen

Resolution= the smallest separation distance at which 2 separate structures can be distinguished from one another

Question 6

Explain how to use an eyepiece granule and stage micrometer to measure the size of a structure

Answer 6

1. Place micrometer on stage to calibrate eyepiece graticule
2. Line up scales on graticule and micrometer
3. Count how many graticule divisions are in 100 µm on the micrometer
4. Length of 1 eyepiece division = 100 µm / number of divisions
5. Use calibrated values to calculate actual length of structures

Question 7

Actual size =

Answer 7

image size / magnification

Question 8

Outline what happens during cell fractionation and ultracentrifugation

Answer 8

1. Blend and homogenise tissue to break open cells and release organelles
2. Filter homogenate to remove debris
3. Perform differential centrifugation:
   A. spin the homogenate in the centrifuge
   B. the most dense organelles in the mixture form a pellet
   C. filter off the supernatant sand spin again at a higher speed

Question 9

Describe how optical microscopes work

Answer 9

1. Lenses focus rays of light and magnify the view of a thin slice of specimen
2. Different structures absorb different amount and wavelengths of light
3. Reflected light is transmitted to the observer via the objective lens and eyepiece

Question 10

Describe how a transmission electron microscope works

Answer 10

1. Pass a high energy beam of electrons through thin slice of specimen
2. More dense structures appear darker since they absorb more electrons
3. Focus image onto fluorescent screen or photographic plate using magnetic lenses

Question 11

Describe how a scanning electron microscope works

Answer 11

1. Focus a beam of electrons onto a specimens surface using electromagnetic lenses
2. Reflected electrons hit a collecting device and are amplified to produce an image of a photographic plate

Question 12

State the order of sedimentation of organelles during differential centrifugation
(from most dense to least dense)

Answer 12

Nucleus
Mitochondria
Lysosomes
RER
Plasma membrane
SER
Ribosome so

Question 13

Explain why fractionated cells are kept in a cold, buffered, isotonic solution

Answer 13

Cold: Slow action of hydrolase enzymes
Buffered: maintain constant pH
Isotonic: prevents osmotic lysis: shrinking of organelles

Question 14

What is the role of lysosomes in digesting bacteria

Answer 14

-lysosomes fuse with vesicle (phagosome)
- releases hydrolytic enzymes