preparing samples

Question 1

what are the 4 steps to prepare samples

Answer 1

• fixation
• embedding
• sectioning
• staining

Question 2

what is the fixation step of preparing a sample

Answer 2

freezing the cells and tissues in their natural state to stabilise the cell structure and preserve the tissues shape. it prevents the sample from decaying

Question 3

what are the advantages to fixation

Answer 3

preserves tissue structures, maintains cell components in their natural state, allows storage for longer periods of time

Question 4

what are the disadvantages to fixation

Answer 4

over fixation can mask or change some cellular components

Question 5

what is the embedding step of preparing a sample

Answer 5

after fixation the tissue if soft and fragile so needs to be strengthened so it can be sliced into thin sections. this is done by embedding the tissue in solid mediums, for light microscopy this is something like paraffin wax but for electron microscopy its something more rigid such as resin

Question 6

what are the advantages to embedding

Answer 6

provides support, making thin slicing possible, enables consistent sectioning and better quality slides, relatively quick and inexpensive

Question 7

what are the disadvantages to embedding

Answer 7

can sometimes extract or alter small molecules, resin is time consuming and requires special equipment and skills, improper embedding can cause deformation

Question 8

what is the sectioning step of preparing a sample

Answer 8

thinly slicing to allow light or electrons to pass through

Question 9

how thinly sliced do samples need to be for light microscopes

Answer 9

microtome cuts slices to 4 - 10 micrometers thick.

Question 10

how thinly sliced do samples need to be for electron microscopes

Answer 10

ultramicrotome cuts into thinner slices of 50 - 100 nanometers thick. Electrons require thinner samples to generate detailed images

Question 11

what are the advantages to sectioning

Answer 11

produced the necessary thin slices, enables visualisation of internal structures at different depths. Provide higher resolution

Question 12

what are the disadvantages to sectioning

Answer 12

requires skill and precision, poor sectioning can tear / distort. Too thick or thin sections affect image quality, equip can be expensive and delicate

Question 13

what is the staining step of preparing a sample

Answer 13

adding colour to create contrast by using dyes that bind to elements of the cell. it helps to distinguish between parts of the cell

Question 14

what are the advantages to staining

Answer 14

adds contrast, makes structures visible, highlight cell components, simple and accessible

Question 15

what are the advantages to staining

Answer 15

some stains not specific which causes background colouring or confusion. Overstaining can obscure details, takers time and complexity

Question 16

what is Haematoxylin

Answer 16

dye that binds to acidic substances like DNA and RNA, found in the cell nucleus. stains the nuclei blue or purple, makes them stand out clearly.​

Question 17

what is eosin

Answer 17

stains basic substances, such as proteins in the cytoplasm and extracellular matrix, in pink or red.​