Microscopy and staining

Question 1

Four main microscopes

Answer 1

light:
compound and confocal (laser scanning)

electron:
transmission and scanning

Question 2

Magnification of light and electron microscopes

Answer 2

light - 2000x

electron - 500 000x

Question 3

Advantages of light microscope (5)

Answer 3

Cheaper, smaller, portable
Specimens living or dead
Simple slide preparation
Can see colour of sample (or stain)
Doesn't require vacuum

Question 4

Advantages of electron microscope (2)

Answer 4

Higher magnification
Higher resolution/ Very small resolving power

• images are black and white but can be coloured digitally

Question 5

Resolution of a light microscope - compound

Answer 5

200nm

Question 6

Resolution of a TEM

Answer 6

0.2nm (highest resolution of microscopes, so number is smallest)

Question 7

Resolution of a SEM

Answer 7

5nm - often given as 0.2nm in exam questions, the same as TEM

Question 8

Which microscope produces a 3D image?

Answer 8

Scanning electron microscope

Question 9

Which microscope produces a virtual image using two convex lenses?

Answer 9

Compound light microscope

Question 10

What is resolution? definition

Answer 10

The smallest distance between two particles/lines/points which allows them to be distinguished from one another

Question 11

What happens to an image if magnification is increased but resolution isn’t?

Answer 11

As magnification increases, the limited resolving power gives blurred images

Question 12

What is resolution limited/determined by?

Answer 12

Wavelength of the light

Objective length aperture (larger gives better resolving power)

Question 13

Order of units of measurement

Answer 13

metre, centimetre, milimetre, micrometre, nanometre, picometre

Question 14

Why is staining used?

Answer 14

Images tend to be low contrast as most cells do not absorb lot of light
Cytosol (aqueous interior) of cells and cell structures are often transparent

Question 15

Gram stain technique:
What does it distinguish?
What substances are used?
What are the results?

Answer 15

-distinguishes gram positive and gram negative bacteria
-Crystal violet then iodine is applied. Slide is washed with alcohol.
Gram positive: retain stain so appear blue/purple
Gram negative: lose stain so stained with safranin dye (a counterstain). Bacteria appear red

Question 16

Acid fast technique:
What does it distinguish?
What substances are used?
What are the results?

Answer 16

Differentiates different species of Mycobacterium
Liquid solvent carries carbolfuchsin dye into cells. Cells washed with dilute acid-alcohol solution

Mycobacterium retain carbolfuchsin stain and so are bright red.

Question 17

What type of staining distinguishes between two types of organisms that would otherwise be hard to identify, or between different organelles of a single organism?

Answer 17

differential staining

Question 18

Two positively charged and two negatively charged dyes? Which one stains the cell components?

Answer 18

+ crystal violet and methylene blue
- nigrosin and Congo red

Positively charged dyes stain cell components, negatively charged dyes stay outside of the cell

Question 19

Four types of sample slide preparation

Answer 19

dry mount, wet mount, squash slides, smear slides