2.1,2,3 Microscopy

Question 1

What is the cell theory?

Answer 1

Both plants and animals are made of cells
Cells are the basic unit of life
Cells only develop from existing cells

Question 2

How does a light microscope work?

Answer 2

There are 2 lenses - objective lens and eyepiece lens
Objective lens produces a magnified image, which is magnified again by the eyepiece lens
Illumination from a light underneath the sample

Question 3

Types of sample preparation

Answer 3

Dry mount
Wet mount
Squash slides
Smear slides

Question 4

Dry mount

Answer 4

Specimens are viewed whole or cut into thin slices

Specimen is placed on a slide and a cover slip on top

Question 5

Wet mount

Answer 5

Specimens are suspended in a liquid such as water or immersion oil
Coverslip is placed on from an angle

Question 6

Squash slides

Answer 6

Wet mount is prepared
Lens tissue is used to press down on the coverslip
Squash slides are used to look at soft samples

Question 7

Smear slides

Answer 7

Edge of a slide is used to smear the sample
Creates a thin, even coating on another side
Cover slip is placed over the sample

Question 8

What is bright field microscopy?

Answer 8

Sample being illuminated from below with white light and observed from above

Question 9

What is wide field microscopy?

Answer 9

Whole sample is illuminated at once

Question 10

Why is staining used?

Answer 10

Interior of cells is usually transparent
Increases contrast between organelles
To differentiate between 2 types of organisms

Question 11

What charge is crystal violet and methylene blue?

Answer 11

Positively charged - attracted to negatively charged materials in the cytoplasm, so cell components are stained

Question 12

What charge is Nigrosin and Congo red?

Answer 12

Negatively charged, so they are repelled by negatively charged cytosol. These dyes stay outside the cell, so cells stand out against stained background

Question 13

What is negative staining?

Answer 13

Using negative/acidic stains that do not bind to the specimen
Clear specimen is observed against a dark coloured background
The negative stain does not penetrate the cell surface membrane

Question 14

What is differential staining?

Answer 14

Distinguishing between 2 types of organisms that would be hard to identify otherwise. It can also differentiate between different organelles of an organism

Question 15

What is the gram stain technique?

Answer 15

Used to separate bacteria into 2 groups - gram positive and gram negative
Crystal violent is added, then iodine (fixes the dye)
Slide is washed with alcohol
Gram positive bacteria will retain the crystal violet stain and appear blue/purple
Gram negative bacteria have thinner walls, so will lose the stain
These are stained with safranin dye to turn red

Question 16

Gram positive/negative bacteria and penicillin

Answer 16

Gram positive bacteria are susceptible to antibiotic penicillin - prevents formation of cell walls
Gram negative bacteria have thinner cell walls and are not susceptible to penicillin

Question 17

What is the acid fast technique?

Answer 17

Used to differentiate mycobacterium from other species
Lipid solvent is used to carry cabolfuchsin dye into the cells
Cells are washed with a dilute acid-alcohol solution
Mycobacterium aren’t affected by the acid-alcohol solution and retain the stain (bright red)
Other bacteria lose the stain and are exposed to methylene blue stain

Question 18

Preparing slides

Answer 18

Fixing - chemicals like formaldehyde are used to preserve specimens
Sectioning - specimens are dehydrated with alcohol, placed in a mould with wax/resin to form a hard block, can be slices with a knife called microtome
Staining - sample are treated with multiple stains to show different structures
Mounting - specimens are secured to a microscope slide and a cover slip on top

Question 19

How does electron microscopy work?

Answer 19

Beam of electrons with wavelength less than 1nm illuminates the specimen

Question 20

What are artefacts?

Answer 20

Structures produced due to the preparation process

Question 21

2 types of electron microscope

Answer 21

Transmission electron microscope

Scanning electron microscope

Question 22

TEM

Answer 22

Beam of electrons transmitted through a specimen

Best resolution - 0.5nm

Question 23

SEM

Answer 23

Beam of electrons sent across the surface of the sample and reflected electrons are collected
Resolving power of 3-10nm
Produces 3D images

Question 24

Why is the inside of an electron microscope a vacuum?

Answer 24

Ensures the electron beams travel in straight lines

Question 25

Advantages of light microscope

Answer 25

Inexpensive to buy and operate
Small and portable 
Simple sample preparation 
Natural colour of sample is seen or stains are used 
Specimens can be living or dead

Question 26

Disadvantages of light microscopes

Answer 26

Lower magnification up to x2000

Lower resolution around 200nm

Question 27

Advantages of electron microscopes

Answer 27

Large magnification of over x500 000

High resolutions

Question 28

Disadvantages of electron microscopes

Answer 28

Expensive to buy and operate 
Large and needs to be installed 
Complex sample preparation that could damage sample 
Vacuum is required 
Specimens have to be dead

Question 29

How does a laser scanning confocal microscope work?

Answer 29

A single spot of focused light is moved across a specimen
This causes fluorescence from the components labelled with a dye
The light emitted from the specimen is filtered through a pinhole aperture

Question 30

Why is a laser used instead of light?

Answer 30

This results in higher intensities and improves the illumination

Question 31

How is a 3D image produced in LSCM?

Answer 31

By creating images at different focal planes

Question 32

What is magnification?

Answer 32

How many times larger the image is than the actual object being viewed

Question 33

What is resolution?

Answer 33

The ability to see objects as separate entities

Question 34

What is resolution limited by?

Answer 34

The diffraction of light as it passes through samples and lenses
Diffraction is the spreading out of light waves

Question 35

Magnification formula

Answer 35

Size of image/actual size of object

Question 36

What is an eyepiece graticule?

Answer 36

Glass disc marked with fine scale of 1-100

Remains unchanged for all objective lens

Question 37

What is a stage micrometer?

Answer 37

Microscope slide with a very accurate scale in micrometres engraved on it