Microscopy

Question 1

Resolution

Answer 1

The minimum distance between two objects in which they can still be viewed as separate.

Question 2

Magnification

Answer 2

How many times larger the image produced is, compared to the object.

Question 3

Magnification equation

Answer 3

M= Image size / Actual size

Question 4

4 microscope types

Answer 4

> Light microscope
Transmission electron microscope
Scanning electron microscope
Laser scanning confocal microscope

Question 5

Light microscopes: +

Answer 5

> Can view living samples
Colour image obtained

Question 6

Light microscopes: -

Answer 6

> Poor resolution due to long wavelength of light
Small organelles not visible

Question 7

4 slide preparations

Answer 7

> Dry mounts
Wet mounts
Squash slides
Smear slides

Question 8

Dry mounts

Answer 8

Thin slices or whole specimens are viewed with just the coverslip placed on top

(plant tissue or hair)

Question 9

Wet mounts

Answer 9

Water is added to the specimen before lowering the coverslip with a mounted needle to prevent air bubbles forming

(aquatic organisms)

Question 10

Squash slides

Answer 10

Wet mounts which you push down on the coverslip to squash the sample to ensure it is thin enough to enable light to pass through

(root tip squash - chromosomes in mitosis)

Question 11

Smear slides

Answer 11

Using the edge of another slide to smear the sample across another slide to create a smooth, thin, even coated specimen, before adding coverslip

(examining blood cells)

Question 12

Eyepiece graticule

Answer 12

> Scale on a glass disc
Used to measure the size of objects being viewed
Must be calibrated each time objective lens (magnification( is changed
It is calibrated to work out the distance between each division presented

Question 13

Calibrating an eyepiece graticule

Answer 13

1) Line up stage micrometer and eyepiece graticule while looking through the eyepiece
2) Count how many divisions on the eyepiece graticule fits into one division on the stage micrometer
3) Each division on the micrometer is 10um, so divide 10 by the number of eyepiece divisions to find what one eyepiece division is at that magnification

Question 14

Differential staining:

Answer 14

> Uses chemical stains to stain different parts of a cell in different colours
Staining improves resolution, visibility, and contrast

Question 15

Crystal violet and Methylene blue

Answer 15

> Positively charged
Attracted to and stain negatively charged material

Question 16

Nigrosin and congo red

Answer 16

> Negatively charged
Cannot enter cells (repelled by cytosol)
Creates a stained background - unstained cells stand out

Question 17

Gram staining

Answer 17

> Used to visualise different bacteria
Uses crystal violet and safranin

Question 18

Gram staining process: Gram positive

Answer 18

> Crystal violet is added
Iodine is added to fix the stain
Alcohol is added to wash away unbound stain
Gram-positive bacteria appear blue/purple as stain is retained due to thicker peptidoglycan cell wall absorbing due

Question 19

Gram staining process: Gram negative

Answer 19

> Gram-negative bacteria cannot absorb crystal violet due to thinner peptidoglycan cell walls
Safranin is added as a counterstain, turning them red

Question 20

Importance of gram staining

Answer 20

> Distinguishing between the bacteria allows prescription of appropriate antibiotics to patients

(e.g. penicillin can treat gram-positive bacteria but not gram-negative)

Question 21

Rules of scientific drawings

Answer 21

1) Pencil
2) Title the specimen
3) State magnification
4) Annotate cell components, cells, and sections of tissue visible
5) Use solid lines, no overlapping

Question 22

Electron microscopes: +

Answer 22

> Uses a beam of electrons (short wavelength) focused by an electromagnet
High resolution

Question 23

Electron Microscopes: -

Answer 23

> Samples must be in a vacuum
Only views non-living specimens
Non-coloured image - samples must be stained

Question 24

Transmission electron microscopes (TEM)

Answer 24

> Extremely thin specimens stained and placed in vacuum
Beam of electrons is transmitted through the specimen
Some parts of the specimen absorb the electrons, and appear darker
2D image
Detailed image of internal structure of cells

Question 25

Scanning electron microscopes (SEM)

Answer 25

> Specimens do not have to be as thin
As electrons are beamed onto the surface and scatter across the surface contours
3D image

Question 26

Laser scanning confocal microscope

Answer 26

> Fluorescent microscope
Uses vey high light intensity to illuminate the specimen stained with fluorescent dye
High resolution
High depth selectivity
Enables viewing of sections of tiny structures that would be challenging to physically section off
Point-by-point focused laser beam (2D)
Use of different focal planes (3D)
Light is emitted from the specimen causing fluorescence