Lecture 2: Fixation Flashcards
what is the definition of fixation
A process by which the constituents of the cells or tissues are fixed in a physical and chemical state (preserved) so that they will withstand subsequent treatment with various reagents with a minimum loss, distortion or decomposition.
what are the aims of fixation
- To preserve cells and tissue constituents in a state identical to that existing in life. i.e. To preserve tissue. 2. Prevention of autolysis(self-digestion) 3. Prevention of putrefaction(decomposition, rotting) 4. Protect tissue from subsequent processing
- define autolysis and putrefaction
Autolysis - shutdown of Oxygen supply (prevents enzyme activity) and the subsequent break down (cell lysis)of living tissue. Putrefaction - the breakdown of the tissue by micro-organisms.
- what are the principles of fixation
Fixation results in denaturation and coagulation of tissue
- Stabilises proteins in the tissue via cross-link formation between soluble proteins and structural proteins which mechanically strengthens the structure and renders it insoluble.
- Reduces solubility of proteins via precipitation and coagulation
- Gel is formed
- list 3 types of fixation
- Heat-denatures and coagulates proteins -> some distortion
- Cryostat (Freezing)-does not denature protein -> minimises distortion.
- Chemicals –chemical fixatives are used
- Perfusion
- Immersion
- define simple fixative
Simple: comprising just one component. e.g.
Ethanol
Acetone
Chromic acid
Picric acid
- what is an ideal fixative
An ideal fixative :
- should be non-toxic,
- non-flammable
- readily available
- cost effective.
- should not interfere with subsequent processing
- should not compromise end result.
What properties should an ideal fixative have?
- Good tissue penetration
- Preserves cellular constituents
- Prevents fixation artifacts
- Prevents structure deformation-maintaining shape and volume
- Stabilisers tissue, preserving the character and distribution of cellular components
- Avoid excessive hardness of tissue
- Allows enhanced staining of tissue
- Safe to handle
- Formalin fixative is best for demonstrating lipids. T or F?
False
- list 5 factors affecting fixation
- pH of fixatives –buffering achieves a desired pH. e.g.. Phosphate salts
- Temperature–used with caution
- Specimen size-penetration rate differs
- Volume changes (Osmolarity)-Cell volume changes because of the membrane permeability
- Concentration of fixative -used with caution
- Duration of fixation process-used with caution
- what effects does lowering and increasing temp have on tissue fixation
Most fixation protocols performed at room temperature.
Lower temperatures (0-4℃)
- Choice for EM and histochemical studies
- Autolysis slowed down
- Diffusion of cellular components slowed down
Higher temperatures (60℃)
- Fixation reactions faster at higher temp = more rapid fixation
- Useful on occasion of urgency
- High risk of morphology distortion
- what are rates of diffusion dependent on
- Temperature (higher the temperature, faster the fixation rate)
- Specimen size (The smaller it is the faster the penetration rate)
- Fixation time and type of fixative:
- d = K√t
- K value is specific for each fixative and affected by the density of the tissue
- Adequate fixation time can vary between different types of fixative.
- list some methods of enhancing fixation for large specimens
- Large and/or dense specimens should have their fixative replaced daily to optimize the benefits of fixation and to prevent the formation of formalin artifacts (effective concentration of formalin diminishes over time)
- Slicing longitudinally so that there is exchange of fixative with the tissue
- what are the effects of osmolarity on cells
- Hypertonic solutions have higher salt content than inside the cell and result in cell shrinkage.
- Isotonic solutions have the same solute concentration as fluid inside the cell
- Hypotonic solutions have a lower salt content than inside the cell and result in cell swelling.
- Define the word fixative
A fluid (mixture of several reactive chemicals) into which the sample is placed so that by processes such as:
- denaturation
- cross-linking of proteins
autolysis is prevented, the specimen is hardened to withstand further processing, and the specimen is preserved in a close living like state in regard to cellular morphology.
- Prolonged exposure of specimens to formalin assist in better fixation. T or F? Why
False
Prolonged exposure introduces artefacts (heme pigment), damages the tissue and causes excessive hardening to the tissue.
- what are the effect of poor fixation
- Cellular integrity of tissue is compromised
- Tissue morphology compromised
- Artefacts/pigments introduced
- what are limitations of fixatives
- Limited section thickness
- Loss of tissue constituents i.e. lipids and enzymes
- Slow procedure
- Limited use on hard tissues
- Is not appropriate for:
a. Formaldehyde induced fluorescence
b. Autoradiography
- name some artefacts found in tissue caused by fixation
- Formalin - heme pigment – brown/black deposit formed in acid formalin particularly after prolonged immersion
- Mercury pigment artefacts – uniform black granular deposit which can result from the use of a primary or secondary fix in mercury contained fixative
- Dichromate – removed by washing in water
- what are the 3 classifications of fixation
I. Microanatomical (histological), preserves the anatomy of the tissue)
II. Histochemical (chemical composition of the cells and tissues of the body)
III. Cytological (the formation, structure, and function of cells, preserve intracellular structure of cells)
- what is difference between simple and compound fixative and give an example of each
Simple - comprising just one component.
- Ethanol, Acetone, Chromic acid, Picric acid
Compound - comprising an empirical mixture of components.
Most microanatomical fixatives are compound fixatives combining one or more fixative so that the disadvantage of one is reduced by the advantage of another fixative.
- NBF, Bouin’s fluid, Mercury (B5)
- list how fixative can affect tissue
-
- what are the 2 buffer salts in NBF
- Sodium dihydrogen phosphate
NaH2PO4
- Disodium hydrogen phosphate
Na2HPO4
- What is the pH of NBF and why is this relevant to histological studies?
pH 7
relevance: fixing tissue in a state similar to life
- What are the advantages/disadvantages of using methanol and acetone as fixative
What are the advantages/disadvantages of using Formaldehyde fixatives (e.g. 10% neutral buffered formalin)
- What are the advantages/disadvantages of using; mercury
- What are the advantages/disadvantages of using; alcohol
- What are the advantages/disadvantages of using; Picric Acid
- What are the advantages/disadvantages of using; dichromate
Modes of Action: What do these fixatives do to the tissue?
- Formalin
- Mercury
- Alcohol
- Picric Acid
- Dichromate
