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clearing agent
Impregnation (infiltration) o Recommended for urgent biopsies
Removal of clearing agent and replaced by a o Gives the fastest result
medium that will completely fill all the tissue o
cavities Factors Affecting Paraffin Wax Impregnation
1. Nature and size of tissue
Embedding (casting or blocking) 2. Type of clearing agent used
Process by which the impregnated tissue is placed Benzene and xylene easily
into a precisely arranged position in a mold removed
containing a medium which is then allowed to Chloroform and cedarwood oil
solidify difficult to remove
Substitute for Paraffin Wax
Embedding media: 1. Paraplast melting point of 56-57C, more elastic
1. Paraffin wax and resilient
2. Celloidin 2. Embeddol melting point of 56-58C, less brittle
3. Gelatin and less compressible
3. Bioloid recommended for embedding eyes
PARAFFIN WAX IMPREGNATION 4. Tissue Mat contains rubber
Paraffin 5. Ester Wax melting point of 46-48C, can be used
Simplest, most common, best embedding medium for impregnation without prior clearing
Melting point: 54-58C at 20-24C, 50-54C at 15- 6. Water Soluble Wax does not require
18C dehydration and clearing
Advantages: melting points 38-420C or 45-560C
1. Thin sections cut easily without distortion
2. Rapid processing CELLOIDIN IMPREGNATION
3. Tissue blocks can be stored in paraffin for an Celloidin
indefinite period of time without tissue distortion - purified form of nitrocellulose soluble in many
4. Good result staining solvents
Disadvantages: - suitable for large hollow cavities
1. Overheated makes tissue brittle Advantages:
2. Prolonged cause excessive tissue shrinkage 1. Permits cutting of thicker tissues
3. Inadequate promote retention of clearing agent 2. Rubbery consistency to be cut without undue
tissue soft, shrunken distortion
4. Difficult to in filtrate 3. Helps to soften brittle layers of dense tissues
5. Paraffin not for fatty tissues 4. Does not require heat when processed
Disadvantages:
560C = normally used for routine work 1. Very slow
2. Very thin sections are difficult to cut
Three ways of Paraffin Wax Impregnation 3. Serial sections difficult prepare
Manual Processing 4. Vapor of ether very in flammable
o At least 4 changes of wax at 15 minutes 5. Photomicrographs difficult to obtain
interval each 6. Very volatile
o 3 hours impregnation time
Automatic Processing Two Methods for Celloidin Impregnation of tissue
o 2-3 changes of wax, decreased processing 1. Wet Celloidin Method - recommended for bones,
time because of constant agitation teeth, large brain sections & whole organs
o Makes use of an automatic tissue Process:
processing machine Fixation -> (12-24hours)
o Fixes, dehydrates, clears and infiltrates Thin, medium celloidin -> (5-7 days)
tissue Thick celloidin -> (3-5 days) stored in 70% alcohol
o E.g. Autotechnicon, Elliot Bench Type
Processor 2. Dry celloidin Method whole eye sections
Vacuum Embedding - same Principle except that of 70% Alcohol, it is
o Negative atmospheric pressure inside the GILSONS MIXTURE (equal parts of chloroform
embedding oven and cedarwood oil)
- Best stored in air-tight jar o Used in hard tissues such as undecalcified bone
and for high resolution light microscopy of
NITROCELLULOSE METHOD tissue sections thinner than usual 4- 6 um.
Low Viscosity Nitrocellulose o Plastic are classified as: epoxy, polyester and
- Equal concentration of ether and alcohol, with acrylic
lower viscosity o EPOXY: are made up of a carefully balanced
- Used in higher concentrations and still penetrate mixture of epoxy plastic, catalysts and
tissues rapidly accelerators.
3 types:
- More explosive handle with care
Bisphenol A (araldite)
Glycerol (Epon)
GELATIN IMPREGNATION
Cyclohexene dioxide (spurr)
Used as an embedding medium for delicate Disadvantages:
specimens and frozen tissue sections because it Hydrophobic
prevents fragmentation of tough and friable Reduce antigenicity
tissues Compromise the result of
Fixation -> 10% gelatin with 1% phenol (24H) immunohistochemistry staining
transferred to 20% gelatin with 1 % phenol (12H) Vinylcyclohexane dioxide carcinogenic
20% gelatin with 1 % phenol
o POLYESTER: were originally introduced
cooled to refrigerator; transferred to 10%
for electron microscopy.
formalin (12-24H) o ACRYLIC PLASTICS: are made up of esters
Volume of impregnating medium should be at of acrylic of methacrylic acid.
least 25 times the volume of the tissue Used extensively for light
EMBEDDING microscopy
After impregnation, the tissue is placed into a mold Polyglycol methacrylate (GMA) *hydrophilic* and Methyl
containing the embedding medium and this medium Methacrylate widely used because of its hardness as the
is allowed to solidify ideal embedding medium for undecalcified bone
Oil Red O
Fixation : Fresh frozen
Sections : 5um mount on Superfrost
Fat : Brilliant red
Nuclei : Blue