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BSM4101: INTRODUCTION TO MICROTECHNIQUES, Chapter 2: Tissue Fixation -…
BSM4101: INTRODUCTION TO MICROTECHNIQUES
Definition
Histology
Study of cellular organisation of body tissues and organs
Study of relationship between these structures and physiological functions
Microtechnique
Preparation of microscope slides from living materials
Use of microscope
Magnify small objects, observe an object at cellular level, allowing scientist to see their shape of a cell, its nucleus, mitochondria and other organelles.
Two levels
Fine structure
distinguished at level of light microscopy
Ultrastructure
detailed structure of cell cytoplasm, organelles and membranes that not visible using a light microscope
Electron microscope extend details at which subcellular structure can be studied.
Applications of histological studies
Education
help students to learn about microstructure of human, animals and plants
Forensic investigation-autopsy
Identify possible cause of death
Medical-diagnosis for treatment
tissues taken from patient can be study in detail
Learn more about patients conditions
Archeology
Provides information about ancient history
Plant systematics
study about plants
Biopsy vs Microarray
Biopsy
one case per slide
Very expensive and slow for large cohorts
Requires a large amount of reagent
Defintion: procedure to remove a piece of tissue or a sample of cells from your body so that it can be tested in a laboratory
Microarray
numerous pathological samples on one single slide
Very rapid results, even when dealing with large cohorts
Requires less than 1ml total reagent volume for the entire cohort
Definition: used to detect the expression of thousands of genes at the same time
Chapter 2: Tissue Fixation
Purposes
Allow them to undergo further preparative procedures without change
Prevent autolysis and bacterial attack
Preserve cells and tissue component in a life-like state
Fix tissues so they will not change their volume and shape during processing
Factors affect fixation
Volume
Fixative to tissue volume is 20:1
If frequent changes of fixative, lower volumes can be used to prevent exhausted
If volume is insufficient it become acidic
Agitation will enhance the process by ensuring that fresh fixative
solution is constantly washing over the surface of the tissue.
Temperature
Lower temperature significantly to 4 degree, decreases rate of fixation
increase temperature, increase speed
Specimen size; too thick fixative wont penetrate
Concentration
Too high; lead to hardening of the tissue and the
formation of excessive artefacts.
Too low; allow exhaustion of the fixative before the
process is completed.
affect the rate of fixation and the total penetration of fixative
Buffering
pH between 6-8 to prevent extremes acidity or basicity
Phosphate, bicarbonate, cacodylate, and
veronal
Time Length
too short, penetration of fixative and crosslinking of
macromolecules will not be adequate
Longer fixation time may cause over-cross-linking,
making the sample brittle.
Differentiate various types of fixatives and fixing agent
Physical
Cryofixation
Sample need to be frozen quickly or else freeze artifacts may be introduced
Faster freezing process, smaller ice crystal formation size and amount
Preseve the enzyme activity and antigenicity
Artefacts; holes when thawed and quite evident microscopically
Arrest cell activities instaneously at low temperature (-180)
Microwave fixation
Microwave excites molecules to rotate and speed up chemical reactions
Reduces time to less than 20mins compared to 21 hours using conventional processing
Advantages
Minimises artifacts
Can overcome deficiencies using chemical fixation; slow penetration rate
Rapid immobilization
Disadvantages
Harsh
Procedures and instrumentation can be technically challenging
Chemical; require fixative which composed of fixing agents and vehicles
Vehicle; buffer solution such as inorganic salt (NaCl)
maintain constant pH
Compatible with other components of fixative and stains
optimize fixation
Adv; fcan fix large specimens
Classifications
Non-coagulants fixatives
Other fixatives
Coagulants/dehydrant fixatives
Change in tertiary structure; reduce solubility
Ethanol, methanol, acetone
Remove water from tissue; destabilising hydrogen bonding
Alcohol cause shrinkage of tissue
Disadvantages; slow rate of penetration
Qualities of a good fixative
Destroys microorganisms
Extracts inactivated autolytic enzymes
Prevents structure deformation, maintaining shape
and volume
Increases tissue consistency
Prevents fixation artefacts
Maintains its chemical composition
Stabilizes tissue, preserving character and distribution
of cellular components
Good tissue penetration
Cheap, non-toxic, non-flammable, nonirritant