SAS 5: Infiltration, Embedding, and Microtomy Flashcards
It is the process of completely removing the clearing agent and it will be replaced by a medium that will completely fill all the tissue cavities
Impregnation (Infiltration)
This allows easier cutting and handling of tissue sections
Impregnation (Infiltration)
Types of impregnating and embedding media:
- Paraffin wax
- Celloidin
- Gelatin
- Plastic
It is the simplest, most common and best embedding medium used for routine tissue processing.
Paraffin wax
It is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. It is solid at room temperature but melts at temperature up to about 65 or 70C
Paraffin wax
Advantages of Paraffin Wax:
- Serial sections are cut with ease and majority without undue distortion
- Very rapid, allows sections to be prepare within 24 hours
- Tissue blocks and unstained can be stored in paraffin for an indefinite period of time.
- Many staining procedures are permitted
- Nucleic acids (DNA or RNA) may be recovered from them decades after fixation
Disadvantages of Paraffin Wax:
- Overheated paraffin makes tissue brittle
- Prolonged impregnation will cause tissue shrinkage
- Inadequate impregnation promotes clearing agent retention
- Tissues difficult to infiltrate need long immersion for proper support
- Not recommended for fatty tissues
Paraffin wax melts in ____ or an ____
oven or an incubator
Melting the paraffin wax in oven or incubator should be regulated at _____
55-60C
The most common waxes melting point
56C
3 ways of impregnation and embedding of tissues
- Manual processing
- Automatic processing
- Vacuum embedding
For manual processing of impregnation, 4 changes of wax for _____ each interval
15 minutes
This methos makes use of an automatic processing machine (autotechnicon)
Automatic processing
Fixes, dehydrates, clears and infiltrates tissue automatically, thereby decreasing the time and labor needed during the processing of tissues
Automatic processing
It change 2 to 3 wax that is required in removing the clearing agent
Automatic processing
How many processing steps does the Autotechnicon have?
12
How many 1-liter glass beakers does the Autotechnicon have?
10
How many thermostatically wax baths does the Autotechnicon have?
2
It involves wax impregnation under negative atmospheric inside an embedding oven. It reduces the time when tissues are subjected to high temperatures thus minimizing heat-induced tissue hardening
Vacuum processing
It facilitates complete removal of transition solvents, and prolongs the life of wax by reducing the solvent contamination,
Vacuum processing
It hastens the removal of air bubbles and clearing agent from the tissue block, thereby promoting a more rapid wax penetration of the tissue
Vacuum
This technique is recommended for urgent biopsies, for delicate tissues such as lung, brain, connective tissue, bone, eye, and etc.
Vacuum processing
With this technique, time is reduced by 25%-75% of the normal time required
Vacuum embedding/processing
The vacuum pressure should not exceed ____
500 mm/hg
Factors affecting paraffin wax impregnation:
- Nature and size of the tissues
- Type of clearing agents
- Paraffin wax must be pure
- Paraffin may be use twice only
- Fixed microtomes, a relatively hard wax with a higher melting point is recommended
- Heavier microtome knives require harder paraffin wax than lighter ones
Paraffin must be pure and free from:
dust
water droplets
other foreign materials
Substitutes for paraffin wax:
- Paraplast
- Ester wax
- Water soluble waxes
It is highly purified and synthetic plastic polymers
Paraplast
More elastic and resilient than paraffin
Paraplast
It is a wax that is for large dense tissue blocks like bones and brain
Paraplast
It prevents ice crystals formation
Paraplast
It is soluble in common clearing agents, no deposits after staining, and has the same time schedule like paraffin
Paraplast
It is a semisynthetic wax recommended for embedding eyes
Bioloid
It is the product of paraffin, containing rubber with the same time schedule like paraffin
Tissue Mat
This wax is harder than paraffin and is not soluble to water
Ester wax
It can be used for impregnation without prior clearing of the tissue
Ester wax
Heavy duty microtomes used in impregnating with ester wax:
a. Sliding microtome
b. Sledge microtome
Most commonly used water soluble wax
Carbowax
A water soluble wax that does not dehydration and clearing
Carbowa
It does not remove neutral fats and lipids
Carbowax
Suitable for enzyme histochemical studies
Carbowax
Purified form of nitrocellulose
Celloidin impregnation
Suitable for hollow cavities, hard and dense tissues such as bones and teeth
Celloidin impregnation
Suitable for large sections of embryo
Celloidin impregnation
2 Methods of Celloidin impregnation:
- Wet celloidin
- Dry celloidin
A method of celloidin impregnation that is recommended for bones, teeth, large brain sections and whole organs
Wet celloidin
A method of celloidin impregnation that is recommended for whole eyes section
Dry celloidin
Dry celloidin uses Gilson’s mixture, what does it made up of?
made of equal parts of chloroform and cedarwood oil
Another form of celloidin
Nitrocellulose method
It has lower viscosity allowing penetration of the tissue rapidly and makes cutting of thinner sections possible
Nitrocellulose methos
It is rarely used except when dehydration is to be avoided or the tissues are subjected histochemical and enzyme studies
Gelatin impregnation
Prevents fragmentation of tissues when used in frozen section and as embedding medium
Gelatin impregnation
