Lecture 2 - How To Examine Cells And Tissues

Question 1

What is the definition of a Tissue?
(Short definition)

Answer 1

Tissue is a Latin word that means woven

Question 2

What is the definition of a Tissue?
(Long definition)

Answer 2

It is a collection of cells that are adapted to perform a specific function

Question 3

What are the 4 types of tissue?

Answer 3

-Epithelial
-Connective
-Muscle
-Nervous

Question 4

What is the definition of epithelial tissue?

Answer 4

Sheets of contiguous cells, of varied embryonic origins, that cover the external surface of the body and line inertial surfaces including the bodies vessels

Question 5

Give an example of epithelial tissue

Answer 5

The outer layer of the skin
Lining of the small intestine

Question 6

What does connective tissue consist of?

Answer 6

Cells and extracellular proteins/glycoproteins and gels

Question 7

What are the main cells found in connective tissues?

Answer 7

Fibroblasts
Chondrocytes
Osteoblasts/osteocytes/osteoclasts
Stem cells/progenitor cells

Question 8

What are the main products of connective tissues?

Answer 8

Fibres
Ground substance

Question 9

What does ground substance consist of?

Answer 9

Cells
Extracellular matric

Question 10

What are the 3 types of muscle tissue?

Answer 10

Skeletal
Cardiac
Smooth

Question 11

What is the function of nerve tissue?

Answer 11

Fast communication system of the body

Question 12

What is the relationship between millimetres and micrometers?

Answer 12

There are a 1000 micrometers in 1mm

Or a millimetre is x1000 larger than a micrometer

Question 13

What is the relationship between micrometers and nanometers?

Answer 13

There are 1000 nanometers in a micrometer

Or a micrometer is x1000 times larger than a nanometer

Question 14

What is the relationship between millimetres and nanometers?

Answer 14

There are 1000000 nanometers in 1mm

OR a millimetre is x1000000 larger than a nanometer

Question 15

What is the definition for the Limit of Resolution?

Answer 15

The smallest distance by which 2 objects can be separated and still be clearly distinguishable as 2 separate objects

Question 16

Which has better resolving power, light microscopes or electron microscopes?

Answer 16

Electron microscopes

Question 17

What methods are there to obtain a tissue biopsy?

Answer 17

Surgery and then dissection
Scraping methods (curettes, scalpel scrapes)
Sharp needles (needle biopsy, pipelle)
Venupuncture (for blood smears)

Question 18

What methods of tissue biopsy can be used to obtain an endometrial sample?

Answer 18

Scraping (endometrial curettage)
Sharp needle (Pipelle)
Surgery (hysterectomy)

Question 19

What method can be used to obtain a bone marrow biopsy?

Answer 19

Sharp needle

Question 20

Why does a specimen/biopsy require fixing/fixation?

Answer 20

To prevent digestion by microbes or being destroyed by decay

Question 21

What does fixation remove from a specimen?

Answer 21

Water
Stiffens biopsy

Question 22

What is the most common fixative that is used in preparing biopsies?

Answer 22

Buffered formalin solution

Question 23

How can fixation cause biopsy shrinkage and formation of artefacts?

Answer 23

If a specimen is left in fixative for too long it becomes to dehydrated shrinking

Question 24

Once a biopsy has been obtained and fixated, what is the next step?

Answer 24

Embedding

Question 25

What happens in embedding?

Answer 25

Dehydration of specimen in different concs of alcohol Then its immersed in hot paraffin wax which is allowed to solidify

Question 26

What is the normal medium of choice for embedding?

Answer 26

Paraffin wax

Question 27

What happens after embedding has occured?

Answer 27

Sectioning Thinly sliced by microtome

Question 28

What is usually done after a specimen has been sectioned/thinly sliced?

Answer 28

Staining

Question 29

Give some examples of stains commonly used:

Answer 29

Haemotoxyin and Eosin (H&E) Massons trichrome Periodic Acid-Shiff stain

Question 30

What does the Haematoxylin stain part of H&E stain and what to colour does it stain?

Answer 30

Stains nucleus BLUE most strongly

Question 31

What does the Eosin component of H&E stain and what colour does it stain?

Answer 31

Stains alkali stuff (the proteins) Cytoplasm and extracellular matrix stained PINK the most strongly

Question 32

Why is H&E (haematoxylin and Eosin) useful?

Answer 32

Nuclei are clearer and more detailed cytoplasm

Question 33

What is Periodic Acid-Shiff stain commonly used to stain and why?

Answer 33

Kidney Stains anything with sugar attached pink Lots of sugar in kidney

Question 34

What is immunohistochemistry/immunostaining?

Answer 34

Using labelled antibodies which bind to specific tissues/cells so they can be easily examined

Question 35

How does immunohistochemistry allow us to see specific cells or tissues?

Answer 35

A primary antibody that is specific to the antigen found on the specific cell/tissue we want to view binds to the cell of interest A secondary labelled antibody is bound to the primary antibody. This antibody has a coloured dye bound to it which shows the presence of the specific antigen/cell

Question 36

How does immunofluroescence work?

Answer 36

Labelled primary antibody with fluroscent tag attached binds to specific antigen on cell of interest Signal is emitted onto the fluorescent tag, it fluoresces and is visible

Question 37

What is the advantage of Staining specimens?

Answer 37

It creates clear contrast so a specimen can be viewed in detail

Question 38

What is a frozen section?

Answer 38

Surgical specimen on metal disc rapidly frozen Cut with microtome in a freezer Stained with H&E

Question 39

What is the advantages of a frozen section over a Paraffin wax formalin fixed specimen?

Answer 39

Prepared rapidly (used during surgeries) Can be done to fresh tissues

Question 40

What are the disadvantages of a frozen section?

Answer 40

Poor stain retention Do not last very long (months) Poor clarity

Question 41

What are the advantages of a Paraffin wax formalin fixed specimen over a frozen section?

Answer 41

Last forever Clear under microscope Used for diagnosis

Question 42

How are tissues prepared for electron microscopy?

Answer 42

Fixed with Glutaraldehyde Embed in Epoxy resin Stain osmium tetroxide

Question 42

How are tissues prepared for electron microscopy?

Answer 42

Fixed with Glutaraldehyde Embed in Epoxy resin Stain osmium tetroxide

Question 43

What are the differences between light a electron microscopy?

Answer 43

L = normal colours E = monochrome L = cheap easy prep E = Expensive difficult L = living and moving specimens E = Dead and inert L = lower mag and res E = Higher mag and res

Question 44

What does resolution of a microscope depend on?

Answer 44

The wavelength of the radiation it uses

Question 45

Why do electron microscopes have a better/smaller limit of resolution than light microscopes?

Answer 45

They use electrons which have a smaller wavelength than light

Question 46

What are the 2 types of electron microscopy?

Answer 46

TEM (Transition Electron Microscopy) SEM (Scanning Electron Microscopy)

Question 47

What is SEM good for?

Answer 47

Viewing 3D images of the surfaces of specimens

Question 48

What is TEM good for?

Answer 48

Viewing 2D images of Intracellular organelles

Question 49

What is confocal microscopy?

Answer 49

A laser excites a fluorescent dye in specimen Light emitted passes through pinhole to detector

Question 50

What are the advantages of confocal microscopy?

Answer 50

Entire depth of tissue can be examined 3D images can be created Can evaluate and lead to rapid diagnoses of eye diseases

Question 51

What is the advantage of Phase contrast microscopy?

Answer 51

Living cells can be viewed in their natural stated since fixing and embedding is not required

Question 52

What is dark field microscopy?

Answer 52

Illuminating a live sample with light that isn’t collected by the objects lens. This makes the black background with bright objects on it

Question 53

What are the advantages of Dark field microscopy?

Answer 53

Creates an extremely clear contrast Easy to do

Question 54

What stain is best to detect sugars/polysaccharides ?

Answer 54

Periodic acid-schiff reagent

Question 55

What is the simple order of preparing a tissue sample for H&E staining?

Answer 55

Fixation Embedding Cutting/microtomy Staining Mounting

Question 56

What type of histiological technique is likely to be used to identify presence or absence of a tumour?

Answer 56

Haematoxylin and Eosin Staining (H&E)