Microtomy & H&E Staining Flashcards
What is microtomy and why is it used?
Using a microtome to cut tissue into very small sections.
Tissue section is then floated on a water bath and picked up on a slide.
This is used to prepare sections for histological and pathological studies using staining and microscopy.
Describe the different components of a microtome
Tray - keep various things while cutting.
Block holder - where paraffin block is inserted ready for cutting.
Guard - covers a sharp knife.
Knife holder - slide knife in underneath plate.
Waste tray - catches all the flaky wax while cutting.
Thickness display - shows what microtome is set to cut to.
Thickness adjustor
Handwheel lock
Handwheel - lifts block up and down against knife.
Lever for activating handwheel brake
Clamping lever for knife holding base.
Describe the basic process of microtomy
1) Clean block
2) Clamp block
3) Clamp blade
4) Position block
5) Orientate block
6) Face block
7) Section
8) Float out on water bath
9) Mount on slide and label
10) Drain and bake to dry
What is cleaning the block?
Getting rid of any excess paraffin wax around outside of cassette.
Excess wax will prevent block from fitting properly into block holder, so it may come out during cutting.
What is positioning the block?
Bridge gap between block and blade.
Describe what is meant by orientating the block
Adjust some screws to ensure block is completely at right angle to blade.
Describe what is meant by facing the block
Removing half a millimetre of wax, as tissue will be floating a bit above surface of wax before it solidifies in embedding, so it’s not at edge of blade.
Describe the sectioning step of microtomy in ore detail
Block is put back on ice before sectioning.
Section is grabbed with tweezers to support it as the ribbon starts to be produced as the handle turns.
Describe the drain and bake step in more detail
Removing excess water by putting it in a 37 degrees incubator overnight.
Ensure it is completely dry, ready for staining.
What is cryotomy?
preparing thin and frozen sections from fresh tissue using a cryotome.
Describe the process of cryotomy
Fresh tissue is frozen in liquid nitrogen as soon as it comes out of patient.
OCT (embedding medium) is sprayed on surface and fresh tissue is stuck onto OCT.
This holds tissue in place while it’s being cut.
What are the advantages of cryotomy?
Rapid diagnosis - intra-operative (ex. small cell carcinoma or non-small cell carcinoma).
Assessment of margins (ex. Mohs surgery for treating basal cell carcinomas).
Immunofluorescence (ex. blistering inflammatory skin disorders).
Enzyme histochemistry - can’t do enzyme histochemistry on something that has been fixed as fixation prevents enzymes from remaining functional (ex. typing muscle fibres).
Describe the advantages of H&E staining
Demonstrates morphology well.
Gives overall impression of architecture.
Simple protocol.
Easily automated.
What are the progressive and regressive methods f H&E staining?
Progressive method involves adding H&E into tissue gently until desired depth of staining is reached.
Regressive method involves over-staining and then slowly decolourising it with acid/alcohol differentiation.
Describe the process of H&E staining
1) Xylene
2) Alcohol
3) Water
4) Haematoxylin
5) Water
6) Acid alcohol
7) Water
8) Scott’s tap water
9) Water
10) Eosin
11) Water
12) Alcohol
13) Xylene
14) Mounting
15) Microscopy
