Histology Intro Flashcards
1
Q
Define Histology
A
- fundamentally, the processes of histology take a piece of tissue, perform the necessary procedures on it and make it visible under a microscope
2
Q
What is the general work flow of histology?
A
- Fixation= chemical preservation of tissue
- Embedding = solidification of tissue
- Sectioning = cutting of solidified tissue
- Staining = colouring of tissue
3
Q
What are 3 key problems in histology?
A
- Degradation
/Putrefaction/Autolysis - Thickness
- Colour/contrast
4
Q
What are the 2 types of Degradation
A
- breakdown of tissue via unrestricted digestion by its own enzymes
- breakdown via influence of external environment ; light, humiditiy & exogenous microbes
5
Q
How do we prevent degradation ?
A
- via a process called fixing
- most simple = methanol, ethanol & acetone
- works via replacing water in tissues, denaturing protiens by manipulation their hydrophobic interactions
6
Q
Describe how we combat the issue of thickness
A
- physical properties mean tissue is too soft to be cut into sub-mm slices
- tissue needs to be solidified
7
Q
What are the 3 common techniques used to combat thickness?
A
- freezing = uses rapid cooling to set tissue by freezing the aqeuous components solid
- paraffin/wax = sections are secured in block of wax
- resin/plastic = polymer such as GMA embeds tissue in solid polymerised block
8
Q
What are some pros of freezing?
A
- fast & cheap
- maintains good molecular integrtiy due to quick tissue processing & minimal chemical interactions
9
Q
What are some cons of freezing?
A
- tissue architecture may be compromised
- skills required to produce a continous frozen section as opposed to smear
10
Q
What are some pros of wax?
A
- preserves good tissue architecture
- easy to section once embedded
11
Q
What are some cons of wax ?
A
- heating step involved to melt wax can destroy proteins of interest
- sample requires ‘dewaxing’ before use
12
Q
What are some pros of resin ?
A
- preserves excellent tissue architecture
- lack of heating step to polymerise resin/plastic means protien conformation is well preserved
13
Q
What are some cons of resin ?
A
- time consuming, fairly expensive & often require user optimisation
- infiltrating large tissue segments with resin is difficult - can result in sections which are solid around edges but flaky/crumbly in the centre
14
Q
Describe the process of wax/resin sample preparation
A
- collect a suitable size biopsy
- preserve it using fixation
- remove water from sample by dehydration
- Remove alcohols by clearing
- infiltrate tissue with parrafin or polyer
- allow infiltration media to set during embedding
15
Q
What is used to cut an embedded block?
A
a Microtome
16
Q
Describe a microtome
A
- contains a static blade which a sample is moved up and down against to shave a wafer off each time
- after each wafer the sample moves a step closer to the balde to generate the next wafer
- these steps dictate the sample thickness
17
Q
What can go wrong when sampling using a microtome ?
A
- sections often srtick to one another in water causing folds
- bubbles occur when air is trapped under the section expand during staining
- misc. pieces of tissue from contaminated water
18
Q
How are fozen samples cut ?
A
- on a specialised rotary microtome = cryostat
- blade & sample are housed in a cooled chamber to keep sample frozen
- these sampes are arent floated, they stick to a slide which is gently pressed against them
18
Q
How are liquid samples fixed?
A
- alternative technique called Cytospin is used
- uses a funnel attached to a microscope slide which contains a sample
- assmebly is spun in a centrifuge which polarises the sample into a dot on slide which can be stained
19
Q
What are the main three dyes ?
A
- Periodic acid Schiff - carbs pink
- Hematoxylin & Eosin - cytoplasm/ECM/ethryrocytes red/pink & nuclei blue/purple
- Millers/Van Gieson - elastic fibres black, cytoplasm& muscle yellow & collagen red
20
Q
Describe Periodic Acid Schiff
A
- used to demonstrate carbohydrates in tissue sections
- periodic acid oxidises some of the tissue
- this produces aldehyde group, which can then condense with Schiff’s reagent forming a pink colour & demonstrating carbs in the tissue
21
Q
Describe Haematoxylin & Eosin
A
- non-specific stain that highlights gross tissue morphology & architecture
- often go to stain
- stains nuclei blue
- Eosin binds positively charged resisdues & stains cellular & extracellular contents to varying degrees of pink
22
Q
Describe Millers Van Gieson Stain
A
- mixture of picric acid & acid Fuchsin
- small picric acid dye penetrates entire tissue
-larger acid Fuchsin acid displaces the picric from collagen - cytoplasms is yellow & collagen fibres are a deep pink/red
- illers stain adds by staining elastic fibres black/deep purple
