MODULE 2 - MICROSCOPES AND STAINING

Question 1

Laser Scanning Microscopes

Answer 1

> can be used for living samples
produces a high contrast
can take images at different depths to create 3D images
used mainly in medicine + biomedical research
max resolution = 100 nm
max magnification = 1000 + 1000<

Question 2

how is a sample prepared for a Scanning Electron Microscope?

Answer 2

sample has to be coated with metal + viewed in a vacuum

Question 3

can Transmission Electron Microscope produce 3D images

Answer 3

no

Question 4

what is the max resolution of a light Microscope

Answer 4

200nm

Question 5

what is the max magnification of light microscope?

Answer 5

x 1500

Question 6

can light microscopes see coloured images?

Answer 6

yes

Question 7

can can light microscopes view live samples?

Answer 7

yes

Question 8

are light microscope images 2D or 3D?

Answer 8

2D

Question 9

advantages of light microscopes

Answer 9

small, cheap, easy to use

Question 10

max resolution of an electron microscope

Answer 10

0.2nm

Question 11

max magnification of electron microscopes?

Answer 11

from 1,000,000 to many millions

Question 12

can electron microscopes view live images?

Answer 12

no

Question 13

can electron microscopes produce coloured pictures?

Answer 13

no

Question 14

how would you prepare a sample for a TEM

Answer 14

> samples have to be treated with a chemical fixative + sliced into very thin pieces

Question 15

which electron microscope has a higher resolution?

Answer 15

TEM

Question 16

with which electron microscope can you see internal cell structures?

Answer 16

TEM

Question 17

how many times smaller is a nanometer then a micrometer?

Answer 17

1000

Question 18

how many times smaller is a micrometer than a millimetre?

Answer 18

1000

Question 19

how many times smaller is a millimetre than a centimetre?

Answer 19

10

Question 20

how large is each division on the stage micrometer?

Answer 20

1mm

Question 21

how big is each small division on the stage micrometer?

Answer 21

100 micrometers

Question 22

when calibrating a microscope using the stage micrometer and the eyepiece graticule, what calculation do you do?

Answer 22

no. of micrometers on the stage micrometer, divided by the number of divisions on the eyepiece graticule

Question 23

why do we use differential staining?

Answer 23

to distinguish between different microorganisms or cellular structures, allowing for classification

Question 24

what is the first step in staining?

Answer 24

fixation

Question 25

why do we fix specimens before staining?

Answer 25

to preserve their structure and prevent it washing away during staining

Question 26

how are two ways you could stain a specimen? give examples

Answer 26

- with heat e.g. flame fixing of bacteria - chemically e.g. using methanol

Question 27

what is the second step of staining?

Answer 27

actually staining the specimen

Question 28

what is the third step of staining?

Answer 28

rinsing

Question 29

why do we rinse specimens after staining?

Answer 29

to prevent over staining and remove excess stain

Question 30

what is a fourth optional step of staining?

Answer 30

counterstaining

Question 31

why do we sometimes use counterstaining?

Answer 31

to provide additional contrast e.g. gram staining

Question 32

what is the final stage of staining?

Answer 32

drying

Question 33

what positively charged dyes can be used in bacterial gram staining?

Answer 33

methylene blue, crystal violet

Question 34

how to positively charged dyes stain cells?

Answer 34

they are attracted to the negatively charged cell cytosol, leading to the staining of cell components

Question 35

what negatively charged dyes can be used in bacterial gram staining?

Answer 35

congo red, nigrosin

Question 36

how do negatively charged dyes work in gram staining?

Answer 36

they are repelled by the negatively charged cytosol, therefore staining the background, but leaving the cell unstained. makes the cell stand out

Question 37

what are the four stages of gram staining?

Answer 37

- sample stained with positive stain - dye is fixed with iodine - slide is washed with alcohol - counterstain added

Question 38

what colour do gram negative bacteria appear after staining?

Answer 38

pink

Question 39

what colour do gram positive bacteria appear after staining?

Answer 39

blue/purple

Question 40

how do gram positive and gram negative bacteria stain differently?

Answer 40

gram positive bacteria have a thicker peptidoglycan cell wall meaning they retain the dye easier