3. Principles Of The Methods Of Cytological And Histological Examination

Question 1

Medical applications of cytological and histological examination?

Answer 1

Study of cancerous cells
Study of parasites
Genetic analysis diagnosis 
Development of new drugs 
Vaccination creation

Question 2

Brief step to step and in depth explanation of examination ?

Answer 2

Removal of tissue 
Fixation
Rinsing 
Dehydration 
Clearing 
Infiltration 
Embedding 
Sectioning 
Staining 
Mount

Question 3

What are dyes?

Answer 3

–> used for staining - highlight specific components of tissue sample

–> dyes either basic or acidic - due to ability of form electrostatic linkages w/ charged parts of tissues

Question 4

Basic dyes? Examples. What it stains?

Answer 4

Tissue components with -ve charge

Examples:

• Toluidine blue
• Alcian blue
• Methylene Blue and Hematoxylin
• Sudan III
• Nissl

What it stains:

• Nucleic Acids
• Glycosaminoglycan
• Glycoproteins

Question 5

Acidic dyes? Examples. What it stains?

Answer 5

Tissue components with a +ve charge

Examples:

• Orange G
• Eosin
• Fuschin

What it stains:

• Mitochondria
• Secretory granules
• Collagen
• Cytoplasm

Question 6

Hematoxylin and Eosin?

Answer 6

Most commonly used b/c makes most components of cell easily distinguishable as it hi lights

Basophilic comp - affinity to basic dyes - BLUE
Acidophilic comp - affinity to acidic dyes - PINK

Question 7

Trichromes?

Answer 7

Mallory stain and Mason stain

• Better application than H and E
• They highlight the nuclei/ cytoplasm well –> and distinguish extracellular comp of tissue better

Question 8

Why fixation?

Answer 8

• To have permanently lasting samples
• Stop autolysis: digestion of tissues by enzymes within the cell/other bacteria
• Stop putrefaction: decomposition of proteins
• Stabilise the tissue for further treatment
• Preserve tissue for further treatment

Question 9

What are important steps to be done to carry out fixation?

Answer 9

• Done asap once removed from body = preserve the cells

- Tissues cut to smaller pieces before fixation - faster penetration of fixatives

Question 10

Ways of fixation?

Answer 10

• Chemical

- Physical

Question 11

Chemical Methods? Examples.

Answer 11

• Use fixatives
• Fixatives preserve the tissues:
  • Proteins
  • Nucleic acids
  • Mucosal substances
   The fixatives preserve these thing by making them insoluble
• Chemical fixatives:
  • Cross linking fixatives (non-coagulant)  e.g. FORMALDEHYDE
  • Precipitating fixatives (coagulant)  e.g. ACETONE/ETHANOL
  • Oxidising Agents: e.g. Potassium dichromate
  • Mercurials: e.g. Zenker’s fixative
  • Picrates Fixatives
  • HOPE fixatives

Question 12

Physical Methods? How? Benefits? Disadvantages?

Answer 12

• Carried out by rapidly freezing sections of tissues
• It is then broken into smaller fragments  using cryostat (freezing microtome)
• Then examined

Benefits of Physical Methods:

• Faster
• Gives less exposure to fixatives
• Useful in studying enzymes as it is non-chemical  so will not inactivate them
• Useful in studying lipids as chemical methods (chemical immersion) dissolves lipids

Disadvantages of Physical Methods:

• Lacks morphological detail in comparison to chemical fixation
• Can pose as a biohazard

Question 13

General Embedding and Sectioning?

Answer 13

• After fixation tissues are made RIGID by using embedding substances to form  solid medium
• This occurs to obtain thin sections of tissues to examine
• Thick sections cause issues when looking for target components of the tissue
• Common embedding materials:
  • Paraffin - Only used in light microscopy
  • Plastic resins - Used both light and electron microscopy

Question 14

Paraffin Embedding?
Dehydration?
Clearing?
Infiltration/Embedding?
Trimming/Sectioning?

Answer 14

4) Dehydration:
•Normally carried out by transferring block of tissue through series of alcohol-water solutions
•Water-alcohol solutions begin w/ 50%  to water-free/ absolute alcohol
5) Clearing:
•Alcohol is replaced by solvent (that is readily soluble in alcohol)
•Then the solvent is replaced by melted form of paraffin
6) Infiltration and Embedding:
•When paraffin –infiltrated tissue is placed in fresh paraffin and allowed to cool  embedding begins
•The solvents dissolve the fats of tissues unless fixed with chemicals such as; OSMIC ACID
7) Trimming/ Sectioning: The resulting block is trimmed to expose tissue for sectioning on a microtome

Question 15

Microtome?

Answer 15

USED FOR SECTIONING PARAFFIN-EMBEDDED TISSUES FOR LIGHT MICROSCOPY

Question 16

Staining? Why?

Answer 16

• Staining required b/c most tissue samples = colourless
• Why is done?
  • Highlights the components
  • Allow components to become distinct from one another