INFILTRATION

Paraffin, Celloidin, Gelatin, Plastic resins

Infiltrating Agents

Non-toxic

Odorless and colorless

Capable of flattening after ribboning

Molten between 30-60 degC

Characteristics of Infiltrating agents

Paraffin Wax Infiltration

To do the process, the medium used is PARAFFIN WAX

Paraffin Wax Infiltration

Used in ROUTINE tissue processes and COMMONLY USED

56degC

Melting point of paraffin wax

Compatible with any staining procedures

Allows cutting of serial sections

Advantages of Paraffin Wax Infiltration

Brittle

Use of overheated paraffin can make the tissue _____

excessive hardening and shrinkage

Prolonged process may cause __________

retention of agent

Inadequate process may cause ________

Bones, eyes, brain, teeth

Specimens that are difficult to infiltrate

Twice

Usage of Paraffin wax

Size and type of tissue to be processed

Type of clearing agent used

Use of vacuum embedding

Factors affecting paraffin wax infiltration

Chloroform, Cedarwood Oil

Clearing agents that are difficult to remove from tissue

Xylene, Benzene

Clearing agent that are easily removed from tissues

Manual Method, Automatic Method, Vacuum Method

Methods of Paraffin Wax Impregnation

Manual Method

Used in lab classes

Carried out using paraffin oven

4 changes at 15 mins interval

How many changes of wax and what is the time interval for Manual Method?

55-60 degC / 2-5 degrees above wax melting point

Temperature for Manual Method

AUTOMATIC METHOD

Require use of Autotechnicon

AUTOMATIC METHOD

Tissue transfer type

1-2 formalin, 3-5 increasing ethyl alcohol concentrations, 6-8 xylene, 9-10 melted paraffin wax

Enumerate the formation for tissue processing (12 beakers)

40 degC

Temperature for Automatic Method

2-3 changes

No. of changes required in Automatic Method

2

No. of stations in Automatic Method

Vacuum Method

Most rapid method

Vacuum Method

Infiltration under negative atmospheric pressure inside the oven, fastest

Speed up removal of bubbles and clearing agent

Tissue is less exposed to heat

Advantages of Vacuum Method

Urgent biopsies

Vacuum Method is recommended for?

lungs, brain, eyes, spleen, CNS

Specimens in Vacuum Method

Paraplast

Embeddol

Bioloid

Tissue Mat

Ester Wax

Carbowax

Dimethyl sulphoxide (DMSO)

Substitutes for Paraffin Wax

Paraplast

Mixture of highly purified paraffin and synthetic plastic polymers

Paraplast

Used for bone and brain specimens

56-57 degC

Melting point of Paraplast

Allow easy cutting of serial sections

Give better ribboning of tissues

Blocks are more uniform

Advantages of Paraplast

Embeddol

Similar to paraplast, less brittle

56-58 degC

Melting point of Embeddol

Bioloid

Semi-synthetic wax

Recommended for eye specimens

Tissue Mat

Product of paraffin with rubber, same property as paraplast

Ester Wax

Harder than paraffin and requires heavy duty type of microtome i.e. sliding microtome

Ester Wax

Soluble in alcohol and other clearing agents and because of this we can avoid clearing

Cellosolve

Compatible clearing agent for Ester Wax

3-4 changes

No. of changes required in Ester Wax

Carbowax

A polyethylene glycol which appears solid at

room temp

Carbowax

A water-soluble wax; dehydration and

clearing is avoided

Processing time is reduced

Will not cause excessive hardening, brittleness and shrinkage

Advantages of carbowax

Carbowax

For enzyme histochemistry and study of cytological details

4 changes required

No. of changes required in Carbowax

Difficult to float them out (water solubility)

Disadvantage of Carbowax

Add 10% polyethylene glycol 900

Remedy for the disadvantage of Carbowax

Dimethyl Sulphoxide (DMSO)

Rapid infiltrating agent and will allow thin sections to be prepared

Dimethyl Sulphoxide (DMSO)

with oyster/garlic taste

Celloidin Infiltration

A purified form of nitrocellulose that is soluble in many agents

Colloidon

other name for Celloidin Infiltration

Celloidin Infiltration

For tissues with VERY LARGE CAVITIES that tends to collapse

Neurological specimens, bones, teeth, whole embryo

Examples of tissues with very large cavities

Dense tissues

Type of tissue that is difficult to infiltrate because they easily collapse are supported better

Very slow

Thin sections

Very flammable

Sections for photomicrography are all difficult to obtain

Disadvantages of Celloidin Infiltration

Thin Celloidin

Medium Celloidin

Thick Celloidin

Methods of Celloidin Infiltration

Thin celloidin

2-4%, 5-7 days

Medium celloidin

4-6%, 5-7 days

Thick celloidin

8-12%, 3-5 days

Wet Celloidin Method

Uses 70% alcohol for storage of tissue blocks before staining

bones, brain, teeth, whole organs

Spx in Wet Celloidin Method

Dry Celloidin Method

Uses Gilson’s Mixture for storage

Whole eye specimens

Spx in Dry Celloidin Methods

Chloroform, Cedarwood oil

2 rgts for Gilson’s Mixture

Nitrocellulose Method

Produces harder tissue blocks and allows

cutting of thinner sections

Nitrocellulose Method

Another form of celloidin soluble in equal concentrations of ether and alcohol; highly explosive

use pasticizers (oleum ricini or castor oil)

Remedy for Nitrocellulose Method

Gelatin Infiltration

Carried out for enzyme studies, histochemistry, frozen sections

25x volume of the spx

Required volume in gelatin infiltration

2-3mm

Required thickness for Gelatin Infiltration

Phenol

Prevents molds

harden tissue

Purpose of formalin immersion