Unit 2: Microscopy

Question 1

Define magnification

Answer 1

How much bigger the image is than the sample (specimen)

Question 2

Define Resolution

Answer 2

How detailed the image is, it is the ability of a microscope to distinguish between to points that are close together

Question 3

Equation for magnification

Answer 3

Image size = Actual size x Magnification

Question 4

Two types of microscope

Answer 4

Electron

Light

Question 5

Cell first observed

Answer 5

1665 by Robert Hooke. Observed the structure of thinly sliced cork using an early light microscope. Described the compartments he saw as looking like ‘honeycomb and named these boxes ‘cells’.

Question 6

Two types of electron microscope

Answer 6

Scanning (SEM)

Transmission (TEM)

Question 7

Observed the first living cells

Answer 7

Antonie van Leeuwenhoek constructed a microscope with a single spherical lens in the late 1600s, that magnified up to x275. He was the first person to observe bacteria and protocista in his examinations of pond water

Question 8

Advantages/Disadvantages of TEM

Answer 8

Advantages - Provide high resolution images

Disadvantages- Specimen need to be sliced thinly.

Question 9

Who provided the first evidence for the origins of new plant cells?

Answer 9

Barthelemy Dumortier

Question 10

Robert Brown, an English botanist was the first to observe which structure of a plant cell?

Answer 10

The nucleus

Question 11

Max Resolutions

Answer 11

Light microscope - 0.2 μm
TEM - 0.0005 μm
SEM - 0.003 - 0.01 μm

Lower resolution = better the image quality

Question 12

Max Magnification

Answer 12

Light microscope - x1500
TEM - more than x1,000,000
SEM - less than x1,000,000 but stunning 3-D images

higher magnification the better

Question 13

How does staining enable cell components to become visible?

Answer 13

Resolution is limited by wavelength of light and the diffraction of light as it passes through a sample. As most cell structures are normally transparent, images have low contrast as they do not absorb light. Stains increase the contrast as different components within the cells take up the stain to varying degrees, enabling components to become visible.

Question 14

Give two examples of stains.

Answer 14

Methylene blue - positively charged dye, attracted to negatively charged materials in the cytoplasm - stains RNA/DNA

Congo red - negatively charged dye, that repels negatively charged cytosol. Stains outside of cell, leaving inside unstrained.

Eosin - The most common dye to stain used in histology, stains pink/orange. It is a negatively charged, acidic dye, that binds to basic components of a cell, mainly proteins located in the cytoplasm.

Iodine - Stain commonly used to observe plant cells.

Question 15

What are the rules for producing good scientific drawings?

Answer 15

1. Include a title
2. State magnification
3. Use a sharp pencil for drawings and labels
4. Use white, unlined paper
5. Use as much paper as possible for the drawing
6. Draw smooth, continuous lines
7. Do not shade
8. Draw clearly defined structures
9. Ensure proportions are correct
10. Label lines must be parallel to the top of the page and drawn with a ruler. They must not cross or have arrow heads.

Question 16

Describe electron micrograph images

Answer 16

They are always black and white (but sometimes colour is added artificially to images don’t confuse this with staining).

Question 17

What is the Gram stain technique?

Answer 17

A technique used to separate bacteria into two groups, Gram-positive bacteria (e.g Streptococcus pneumonae - causes pnuemonia) and Gram-negative bacteria (e.g. Yersinia pestis - causes bubonic plague).

Crystal violet is first applied to a bacterial specimen, then iodine, which fixes the dye. Slide then washed with alcohol. Gram-positive bacteria retain the dye where as Gram-negative bacteria have much thinner walls and lose the stain.

Gram-negative bacteria are stained with the counterstain safranin, which makes them appear red.

Question 18

What are the stages involved in preparing stained slides?

Answer 18

1. Fixing - Chemicals like formaldehyde are used to preserve specimens.
2. Sectioning - specimens are dehydrated with alcohols and then placed in a mould with wax/resin to form a hard block that can be thinly sliced.
3. Staining - treatment of specimen with (often multiple) stains to show different structures.
4. Mounting - specimens are then secured to a microscope slide an a cover slip placed on top.

Question 19

What is an eyepiece graticule?

Answer 19

A glass disc marked with a fine scale of 1 to 100. The scale has no unit and remains unchanged whichever object lens is in place. The relative size of the dimensions, however, increases with each increases in magnification. The scale on the graticule at each magnification is calibrated using a stage micrometer.

Question 20

A stage micrometer

Answer 20

A microscope with a very accurate scale in micrometres (μm) engraved on it.