DAVAO DOCTORS COLLEGE

MEDICAL LABORATORY SCIENCE DEPARTMENT

STUDENT NOTES: HPCT

IMPREGNATION
 AKA Infiltration Paraffin Wax Substitutes
 Removal of clearing agent from the tissue and replacing it with 1. Paraplast
the infiltrating media  Mixture of pure paraffin and synthetic plastic polymer
 This infiltrating media will completely fill all tissue cavities, thus (Dimethyl sulfoxide); more elastic and resilient
giving firm consistency, as well as allow easy handling and  MP: 56-57OC
cutting of thin tissue 2. Embeddol – less brittle, and less compressible (MP: 56-57OC)
 Incomplete Impregnation  Airholes in tissue sections 3. Bioloid – semisynthetic; for embedding of eyes
 Main factors: method of impregnation, nature and size of tissue, 4. Tissue Mat – has rubber
clearing agent used 5. Ester Wax
 MP: 46-48OC
Types of Tissue Impregnation and Embedding Media  Harder than paraffin thus used with sliding/sledge-type
1. Paraffin microtome
2. Celloidin (Colloidin)  Water insoluble but soluble in 95% ethanol, thus prior
3. Gelatin clearing is not needed
4. Plastic  But Cellosolve, and xylene may be used if indicated

1. Paraffin 6. Water Soluble Waxes (Polyethylene glycol)

 Simplest, common and best media for routine processing  MP: 38-42OC or 45-56OC
 ADV: sections are cut easily without distortion; very rapid  E.g. Carbowax - most common
(24hrs); permits many staining procedures; o Hygroscopic = absorbs water; no need for
 DADV: not for fatty tissues; must fully impregnate the dehydration and clearing
tissue to avoid tissue crumbling o Easily dissolved in water, thus sections are difficult
 Melting point for routine work: 56OC to float out and mount
o Never overheat (>60OC) – causes brittleness, [Remedy: add soap to water, or 10% polyethylene
shrinkage, hardening; destruction of lymph tissue glycol 900 in water]
 Paraffin oven – maintain at 2 to 5 OC higher than MP of wax o Neutral fats and lipids can be demonstrated
 Used pure o Not exposed to too much heat, thus for enzyme
o Wax must be filtered first using coarse filter paper histochemistry
such as Green’s No. 904 in wax oven at 2OC higher
than MP of wax 2. Celloidin
o Reusable only once, but remove water first by boiling  AKA colloidin
to 100-105OC  Purified form of nitrocellulose/gun cotton
 Specimen with large and hollow cavities which tend to
Methods of Paraffin Impregnation collapse; hard and dense tissues; neurologic tissues
1. Manual  Concentration: in 2%, 4%, 8% dissolved in equal parts of
 Atleast 4 changes of paraffin every 15 minutes ether and ROH
2. Automatic  ADV: Does not require heat for processing; rubbery
 Uses machines like Autotechnicon and Elliot Bench-Type  DADV: very slow (days to weeks)
Processor, which fixes, dehydrates, clears, and infiltrates  Methods:
tissues o Wet Celloidin
 Infiltration is usually at stations 11 and 12  For bones, brain, teeth
 ADV: Has constant agitation  speedy procedure  Procedure:
 NOTE: Any odor in clearing agent indicates that the 1. Fixation & Dehydration
paraffin wax should be changed 2. Place tissue in ether-alcohol
 Wax bath thermostat should be set atleast 3 degrees 3. Thin celloidin
above the MP of paraffin 4. Medium celloidin
3. Vacuum Embedding 5. Thick celloidin
 Fastest (25-75% reduction of usual impregnation time) 6. Remove specimen and the put it in fresh thick
 Uses embedding oven with negative atmospheric pressure celloidin
 rapid removal of air bubbles (e.g. lungs) and clearing 7. Keep in jar or dessicator until ether-alcohol
agent rapid infiltration evaporates
8. Store tissue block in 70%-80% alcohol
 For urgent biopsies, delicate tissues (e.g. CNS, eyes)

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  Complete impregnation: No fingerprint marks on o TissueTek – has paraffin reservoir, tissue
surface of tissue block tank, warm plate and cold plate (-5OC)
4. Disposable o Peel-away (thin plastic mold)
o Dry Celloidin molds o Ice tray (inner mold smeared with
 For whole eye sections glycerin or liquid paraffin),
 Uses Gilson’s mixture (equal parts of chloroform o Paper boats
and cedar wood oil) for storage, instead of 70% to
80% alcohol Other Embedding Methods

o Nitrocellulose Method/Low Viscosity Nitrocellulose
(LVN)
 Has lower viscosity, thus can be used in high
concentrations, and rapid tissue penetration
 ADV: Harder tissue block, thus thinner sections are
possible
 DADV: Explosive when dry d/t nitrates
 Plasticizer (e.g. oleum ricini or castor oil) is needed
to prevent tissue cracking in chrome-mordanted
tissues
3. Gelatin
 Rarely used
 For histochemical, enzyme studies, and frozen sections
 ADV: Water soluble (no dehydration and clearing needed)
 DADV: may decay
 TSE must be <2-3mm thick
 Procedure:
1. Wash out of fixative
2. Put tissue in 10% gelatin with 1% phenol
3. 20% gelatin with 1% phenol
4. Fresh 20% gelatin with 1% phenol
5. Cool in refrigerator
6. 10% formalin
 1% Phenol must be added to prevent molds
 TSE:IMPERGNATING AGENT ratio is set at 1:25
EMBEDDING
 AKA Casting, Blocking
 Placing the impregnated tissue into a mold with embedding
media, and then allowing the media to solidify
 Orientation: Arrangement of the tissue in a precise position
in the mold during embedding
 Surface to be cut should be parallel to bottom of the mold
 Molds should bear the accession number
 Procedure:
o Put tissue with label on a mold, immerse them in melted
paraffin at 5-10 OC higher than MP of wax, then rapidly cool
in a refrigerator at -5 OC, or in cold water
Embedding Molds
1. Leuckhart’s L-shaped strips of heavy brass and metal;
Embedding mold reusable and adjustable
1. Double embedding method– celloidin first, then paraffin; for
large blocks of dense tissues; obsolete
2. Plastic (Resin) embedding
 For High resolution light microscopy of thinner than usual
sections, renal biopsies, BM biopsies
Classes:
o Epoxy
 For electron microscopy
 Most widely applied, but carcinogenic due to
vinylcyclohexane dioxide (VCD) component
 Types:
 Bisphenol A (Araldite) – slow
 Glycerol (Epon) – low viscosity
 Cyclohexene Dioxide (Spurr) – very low viscosity;
fastest
o Polyester
 For electron microscopy; seldom used
o Acrylic Plastics
 For High resolution light microscopy
 E.g., polyglycol methacrylate (GMA), methyl metacrylate
(MMA)
 Benzoyl peroxide – catalyst; forms radicals, which are
site for polymerization
 Acrylic plastics must be stored in dark bottles to prevent
radical formation and premature polymerization
 Embedding media may be stained, thus use
hydrophobic MMA
References:
1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
(2nd Revised Edition). Makati, PH: Katha Publishing
(611.0182/B83)
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
(2015). Basic histopathologic techniques. Metro Manila, PH:
C & E Publishing

2. Compound Interlocking plates resting on flat metal

embedding unit base; for batch embedding
3. Plastic Special stainless steel base mold fitted with
embedding rings a plastic embedding ring (serves as block
and base molds holder during cutting)

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