Histology - Overview and Staining methods Flashcards
what are the 4 main building blocks of life
- proteins
- lipids
- nucleic acid
- carbohydrates
what is histology
a microscopic study of normal cells and tissues, looking at how structure is related to tissue function, and how disease is associated with disruption of normal cell structure and function
what is pathology
a microscopic study of diseased cells and tissues
what are the 4 main tissue types
epithelial
connective
muscle
nervous
what are the two main methods of microscopy
light microscopy and electron microscopy
what is light microscopy
low resolution microscopy used to view basic cell structure
what is electron microscopy
method for viewing the ultrastructure of the cell
what is the microscopy technique used in routine histology
light microscopy
what are the steps to microscopy
- specimen collection
- fixation
- dehydration
- embedding
- sectioning
- staining
- viewing
why are tissue samples cut into thin sections prior to the microscopy
most tissue samples are too thick to be examined directly under a microscope and must first be cut into very thin sections to enable light or electrons to pass through the tissue and make the structure visible
what are the different technique to specimen collection
- incision or a punch biopsy
- needle biopsy
- endoscopic biopsy
what are incision or punch biospies used for
specimen collection from skin or oral surfaces
what are needle biopsies used for
specimen collection from organs or lumps below the skin. to guide the biopsy, imaging techniques like x rays and mris can be used
what does endoscopic biopsy involve
a flexible tube with light and a camera - cutting tools can be used to collect tissue specimens
where would an endoscopic biopsy be used in the body
- trachea
- bronchus
- lungs
- esophagus
- stomach
- small intestine
- colon and rectum
- mainly bladder
where would a needle biopsy be used
the brain
eye
thyroid
lymph nodes
breast
lung and pleura
liver
kidney
bone and bone marrow
testis
skeletal muscle
what kind of biopsy is used for the heart and the liver
transvascular biopsy
what do cells release as they die
enzymes that degrade components for the tissue
what does fixation do
terminates all biochemical reactions and so prevents the release and activity of degradative enzymes as well as inhibiting microbial growth and so prevents tissue decomposition
what are some common fixatives
formaldehyde and glutaraldehyde
why is fixation important during microscopy
it preserves the structural arrangement betwen cells and extracellular components
is paraffin embedding compatible with water
no
how is water removed from tissues during dehydration
using a series of graded alcohols, done gradually to be prevent distortion
why should water not be removed from the tissue sample in a single step
the tissue would shrink rapidly and the structural arrangement would be distorted
why do tissues require embedding
thin tissue sections are delicate and soft and easily distorted, and must be embedded in a hard substance as support
what are most commonly used to embed tissue samples
resin and paraffin
what are tissue samples submersed in prior to embedding and why
resin and paraffin are not compatible with water or alcohol, so after dehydration the sample is then immersed for a period of time in a strong solvent like xylene to replace the alcohol
how should tissue be cut to be viewed under a microscope
thin and flat, ideally just a single layer of cells
why should tissue samples be cut into thin and flat sections
so they are transparent, as this allows the beam of light or electrons to pass through the tissue to enable visualisation
how are embedded speciments sectioned
using a microtome
what is a microtome
a machine that cuts embedded tissues into sections around 7 nanometres thick
what are tissue sections mounted on
glass slides
why are tissue sections mounted on glass slides
for protection
why is staining another important step in microscopy
most cells are colourless and transparent, so staining makes cells and their components more visible
is there a limited number of staining methods available for light microscopy
no there is a wide variety
what does staining allow
identification of different cell and tissue features
what state are most stains for light microscopy
aqueous
are stains compatible with paraffin
no
why are stains not compatible with paraffin
they are aqueous
how do you use stains incompatible with paraffin
reversal of the dehydration steps used earlier, so the embedded sample is first submerged in xylene before then being passed through the graded series of alcohol in order to replace the alcohol with water and leave it ready for staining
does where the tissues are sectioned make much of a difference to tissue architecture
yes
what is the most commonly used staining technique
haematoxylin and eosin (H&E)
what is haematoxylin
a basic dye that stains acidic structures like nucleic acid blue/purple
what is eosin
an acidic dye that stains basic structures such as cytoplasmic proteins red or pink
are epithelial tissues highly cellular
yes - there is very little extracellular material
what is the periodic acid-schiff reaction (PAS)
a method that stains complex carbohydrates magenta
what are mucins produced by
goblet cells
what are examples of PAS-positive molecules
muines produced by goblet cells, brush borders and basement membranes
what is the proximal convoluted tubule
part of the nephron in the kidney. involved in reabsorption of essential nutrients and water to prevent them being excreted within urine
why does the proximated convoluted tubule require a large surface area
it is involved in reabsorption of essential nutrients and water
what does the PAS staining method reveal in the proximated convulated tubule
the prominent brush border projecting into the lumen of the tubule, as well as the basement membrane, as both are strongly PAS positive.
what is the oesophagus
a strong muscular tube that conveys food from the throat to the stomach
how is the mucosa found when in a relaxed state
deeply folded
what do the different colours of masson trichrome represent
the blue purple colour is the nuclei, and other basophilic structures
the green blue is collagen
the red is the cytoplasm, muscle, red blood cells and keratin
explain the immunohisotochemistry technique for staining
utilises antibody specifity for antigen. antibodies are required to identify substances of interest. the antibody is conjugated to an indicator, whether enzymatic or fluorescecnt
what is the most specific staining method
immunohistochemistry
what does immunohistochemistry use antibodies to do
stain specific cellular proteins by using antibodies specific for their antigen to reveal the components by joining the antibody to some sort of colour indicator
what is the alcian blue staining method
- stains mucin and cartilage blue, and can be combined with other stains like H&E
what is the van gieson staining technique
stains collagen red, nuclei and erythrocytes and cytoplasm yellow
what are reticulin fibres components of
connective tissue
what is the reticulin stain
this stains reticulin fibres blue black, and is often combined with H&E
what is the most common form of methods used in histology
light microscopy
how can light microscopy be subdivided
bright field, immunofluorescence and dark field microscopy
give a breakdown of periodic acid schiff
this is a method that stains complex carbohydrates a dark red or magenta. it identifies glycogen in cells
what are the main steps to preparing tissue for bright field microscopy
- fix the tissue using formalin
- process the tissue (remove water with ethanol, remove ethanol with xylene. embed in paraffin - removes xylene)
- section
what happens once the paraffin embedded tissue samples are sliced accordingly
they are mounted on glass slides. staining makes it easier to then see under the microscope and identify the cell components
what is the most common staining method used in bright field microscopy
haemotoxylin and eosin (H&E)
give a breakdown on how H&E staining works
the haemotoxylin is positively charged and stains basophilic structures, which are negatively charged. these include nuclei, ribosomes, cartilage matrix and the rough endoplasmic reticulum
the eosin is negatively charged and stains eosinophilic structures, which are positively charged, pink. these include collagen, cytoplasmic proteins and mitochondria
how does the giemsa stain technique work
this is the standard stain for blood and bone marrow smears
how does alcian blue stain work
this stains acidic polysaccharides blue, like mucin and cartilage
what does van gieson stain
blood vessels and the skin
explain the different staining colours of masson trichrome
stains connective tissue blue
stains nuclei dark red or purple
stains cytoplasm, kertain, and erythro red or pink
what does masson trichrome stain
supporting tissues like collagen
what is PAS
periodic acid schiff
what is the masson trichrome staining technique used for
staining connective tissue - useful for differentiating different types of tissues
