Impregnation or Infiltration is the process of permeating the tissue with a support medium
clearing medium is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities giving a firm consistency to the specimen, and allowing easier handling and cutting of suitably thin sections without any damage or distortion to the tissue and its cellular components
Paraffin Wax Impregnation uses a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils.
simplest, most common
best embedding medium (routine process)
ease in cutting
permanent paraffin blocks
good staining results
not recommended for fatty tissue
Overheated paraffin causes brittle specimen
Inadequate paraffin wax impregnation causes clearing agent retention, where tissue will become soft and shrunken, tissue blocks crumble, break up when floated in water bath
Prolonged paraffin wax impregnation causes tissue shrinkage and hardening
Paraffin wax impregnation is very rapid, done within 24 hrs
May be stored in paraffin for an indefinite period of time after impregnation without considerable tissue destruction
common paraffin wax melting points
56 °C → Routinely used
Melting point of 54-58 °C (wax) at 20-24°C at room temp laboratories
Melting point of 50-54 °C (wax) at 15-18°C laboratory
In paraffin wax impregnation, paraffin oven or an incubation is used which has been regulated at 55-60 °C
Fresh wax should be filtered before use in a wax oven at temperature of 2°C higher than its melting point
solid at room temp but melts at 65 or 70 °C
most common for histological use is 56-58 °C
at melting point tends to be slightly viscous, decreases as temperature increases
to decrease viscosity and improve infiltration technologists increase temp to above 60 or 65 °C
In manual or hand processing in paraffin wax impregnation, the specimen is immersed in another fresh solution of melted paraffin for approximately 3 hrs to ensure complete embedding or casting of tissues.
At least 4 changes of wax are required at 15 mins intervals in order to ensure complete removal of the clearing agent from the tissue
A) 24
B) 6
C) 12
D) 2
E) 1
F) 15
G) 3
Automatic processing in paraffin impregnation uses autotechnicon or Elliott Bench type processor; it has 12 processing steps
decreasing time and labor needed and MORE RAPID and less technicality
Wax bath temperature: 3 °C above the melting point
Dehydration is the most critical step
ADVANTAGE: “Constant agitation”
only 2-3 changes of wax are required to remove the clearing agent and properly impregnate the specimen
Elliott Bench-Type Processor machine is mounted on rollers to permit the turning of platforms and easy access to beakers and wax baths
Vacuum embedding in paraffin impregnation uses “under negative atmospheric pressure” inside an embedding oven
Facilitates complete removal of transition solvents, and prolongs the life of wax by reducing solvent contamination
Recommended for urgent biopsies, delicate tissues such as lung, connective tissues, decalcified bones, eyes, spleen, and CNS.
Use of vacuum embedding gives the fastest results
Larger and denser tissue blocks e.g. bones, fibroids, brains require longer periods and more frequent changes of wax
Benzene and Xylene are easily removed from the tissues
Chloroform and Cedarwood oil are more difficult to remove and require more frequent wax changes
Addition of benzene may hasten displacement of cedarwood oil with less tissue shrinkage
Vacuum embedding
Temperature is maintained at 2-4 °C the melting point of the wax
Time required for complete impregnation is reduced by 25-75% of the normal time
Tissue is not over-exposed to heat; brittleness, shrinkage and hardening of tissues consequent to overheating is therefore prevented
Tissue can also be transferred after clearing to a heated bath of paraffin wax (air can be evacuated)
Infiltration in overheated paraffin >60 °C will produce considerable shrinkage and hardening of tissues
Paraffin oven must be maintained at a temperature of 2-5 °C above the melting point of the paraffin used
Fresh wax should be filtered before use in a wax oven at temperature of 2 °C higher than its melting point
Green’s No. 904 is a coarse filter paper used for the filtration of the “used” wax.
Water must be removed by heating the wax to 100-105 °C (boiling point) thereby to raising its melting point.
Paraffin wax may be used only twice and the paraffin wax must be changed.
Paraplast is a mixture of highly purified paraffin and synthetic plastic polymers
MP: 56-57 °C.
More elastic and resilient than paraffin wax
For large tissue blocks
Blocks obtained more uniform → better ribboning of sections
Preventing formation of ice crystal artifacts
No deposit is left on slides after staining
Soluble in the majority of the clearing agent
Paraplast with a melting point of 56-58 °C is recommended
During the winter = 54-56 °C
May be used if the tissue is cut in a cool room
Summer it may be necessary to use 60-63 °C, avoided if possible in order to not "cook" the tissue.
Cooked tissue does not section well or, if it does, not stain well and most details are destroyed
Embeddol is a synthetic wax; substitute similar to paraplast.
Less brittle and less compressible
MP: 56-58 °C
Bioloid is asemisynthetic wax recommended for embedding eyes
Tissue Mat is a product of paraffin containing rubber, same property as paraplast
Ester wax is harder than paraffin; it is insoluble = not soluble in water but they are soluble in 95% ethyl alcohol
MP: 46-48 °C
Uses Cellosolve or xylene
3-4 changes of wax to ensure complete tissue impregnation
Should be done on a heavy duty microtome
Water soluble wax is mostly polyethylene glycols (plastic polymers)
MP: 38-48 °C or 45-56 °C
Carbowax is most commonly used water-soluble wax.
Soluble in and miscible with water → not require dehydration and clearing of the tissue
Used for enzyme histochemistry studies
Routine processing, 4 changes
Easily dissolved in water → add soap to water or using 10% Polyethylene Glycol 900 = promote flattening and floating out of sections
Polyethylene glycol (18 or more C atoms) appears solid at room temperature
Water soluble wax
Processing time is reduced thus harmful effects produced by ordinary dehydrating agents are avoided.
Does not remove neutral fats and lipids
Soluble in reagents used for routine processing with paraffin, allowing these substances to be demonstrated in thin sections.
Celloidin or Collodion impregnation is recommended for “large hollow organs” which tend to collapse, for hard and dense tissues such as “bones and teeth” for large tissue sections of the whole embryo
Permits cutting of thicker tissue sections → recommended for neurological tissues
Crumbling of tissues avoided
Very slow (several weeks to months)
Thin sections difficult to cut
Serial sections difficult to prepare
Celloidin is a purified form of nitrocellulose soluble in many solvents
Wet celloidin is recommended for bones, teeth, large brain sections and whole organs
Tissue blocks is stored in 70-80% alcohol, to avoid, dehydration and shrinkage of tissues.
Ether and alcohol (equal parts) → 12-24 hours after usual fixation and dehydration
Thin celloidin → 2-4% for 5-7days
Medium celloidin → 4-6% for 5-7days
Thick celloidin (drain and poured)→ 8-12% for 3-5days (until impregnation)
Dry celloidin method is preferred for the processing of whole eye sections
Gilson’s mixture (chloroform and cedarwood oil) is added (before hardening) to the block to make it transparent
Same with bioloid
Principle and procedure: Similar to wet celloidin method, except that 70% alcohol is not used for storage
This method does not make use of alcohol due to the presence of cedarwood oil in the block
Nitrocellulose method is more explosive than celloidin
Tissue to crack → add plasticizers when embedding chrome-mordanted tissue
When dry, striking or dropping the container will cause explosion.
With lower viscosity allowing it to be used in higher concentration and still penetrate tissue rapidly
It is usually marketed while wet with alcohol → container must be kept tightly covered and protected from sunlight to avoid evaporation
Low Viscosity Nitrocellulose has equal concentration of ether and alcohol
Gelatin impregnation is rarely used except when dehydration is to be avoided → histochemical studies
Used for delicate specimen and frozen tissue section
Does not require dehydration and clearing
Low melting point
Tissue not more than 2-3mm thick
1% phenol → prevent the growth of molds
25:1 → ratio of impregnating medium to tissue
Excess gelatin may be removed by floating the sections on to paper and trimming them with scissors