FIXATION – Fixing or preserving fresh tissue for 3.

Thickness and area of section FIXATIVES (ACCDG TO COMPOSITION)

examination  4mm 1. Simple Fixatives: 1 component
Aims of fixation  Electron microscopy: 1-2mm2 substance
 Preserve morphological and chemical  Light microscopy: 2cm2 2. Compound Fixatives: made up of 2 or
integrity of the cell  Fixative placed should be 10-25 times of more fixatives
 Prevent degeneration, decomposition, tissue volume.
putrefaction and distortion of tissues after FIXATIVES (ACCDG TO ACTION)
removal from the body 4. Osmolality of fixative 1. Microanatomical Fixative: General
 Harden and protect tissues from trauma of  Hypertonic: Shrinkage microscopic study
further handling  Isotonic (340mOsm) and Hypotonic: 2. Cytological Fixative; preserve specific
 Properly orients tissue Swelling parts
 Protects medical technologist from  Best obtained in slightly hypertonic soln  Nuclear: ccontains GHAc
infection (400-450mOsm)  Cytoplasmic: No GHAc
 Protein stabilization is the most important  Vehicle osmolality > Total osmolality 3. Histochemical Fixative: preserve
reaction  Glutaraldehyde: vehicle osmolality: chemical constituents
 Preserves by stopping all cellular activities 300mOsm
 Prevents autolysis by inactivating lysosomal PROBLEM CAUSE
enzymes 5. Concentration of fixative Failure to arrest Failure to fix
 Protects tissue from putrefaction  Formalin: 10% early autolysis immediately
 ADDITIVE FIXATION: chemical constituent  Glutaraldehyde: 3% Removal of Wrong choice of
is taken in and becomes part of the tissue,  Glutaraldehyde for immunoelectron substances fixative
cross links, and stabilizes proteins microscopy: 0.25% Presence of artefact Incomplete washing
 NON ADDITIVE FIXATION: agent is not pigments of fixative
incorporated, only alters composition and 6. Duration of fixation Tissues are soft Incomplete fixation
stabilizes by removal of bound water  Prolonged: Shirnkage and hardening, Loss or inactivation Wrong choice of
attached to H-bonds inhibition of different activities and of enzymes fixative
FACTORS INFLUENCING FIXATION reactions Shrinkage and Overfixation
1. Hydrogen ion concentration  Electron microscopy: 3 hours swelling
 Satisfactory at pH6.0 and pH8.0  Specimen be placed ASAP after removal Brittle and hard Prolonged fixation
to prevent autolysis and putrefaction blocks
2. Temperature
 Traditionally carried out at RT 7. Prep of some organs Fixation Artefacts
 Tissue processor: 40C  Uterus and other hollow organs: Formalin Pigment
 Electron Microscopy and Opened and sliced thinly  Produced under acid conditions
Histochemistry: 0-4C  Brain: Suspended in 10% buffd formalin  Eliminated by fix in phenol-
 RNA: 45C 2-3 weeks; prevent flattening by formalin
 DNA: 60C suspending with cord tied under Circle “Crush artefact”
 Urgent biopsy: 60C of Willis
 In surgical specimens, liver biopsies
 Tuberculosis: 100C  Air-filled lungs: cover with several layers
of gauze  Intense eosinophilic staining
 Eyes: not dissected as it may collapse
and wrinkle. Inject formol-alcohol before
fix
FIXATIVE FORMULA/DESCRPXN FIX TIME INDICATION ADVANTAGE DISADVANTAGE PRECAUTIONS
ALDEHYDE FIXATIVES
CH2O /Formalin 10% CH2O (4%) – 24h Used for most tissues  Preserves fat and mucin  Irritating fumes Decomposition to formate
Stock 40% CH2O  Preserves glycogen and  Considerable shrinkage (treat with 10% MetOH) can
diluted protein  Reduces basophilic and ppt white paraformaldehyde
 Does not require washing out eosinophilic staining and (treat with 10%
 Tolerant fixative forms brown precipitates methanol)and brown/black
 Penetrates and does not if unbuffered crystals if present w/ blood
overharden tissue (treat with sat. picrate or 1%
KOH in 80% Alc.)

a. 10% Formol-Saline 40% CH2O diluted to 24h – 35C Microanatomical  Penetrates and does not  Slow fixation
10% with NaCl 48h – 20-25C CNS overharden tissue  Shrinks in alcohol
Post-mortem tissues  Minimum shrinkage dehydration
 Preserve enzymes  Reduced metachromatic
 Demo fat and mucin staining of amyloid

b. 10% 3.5g NaH2PO4 4-24h Preservation and Similar to formol-saline  Time consuming prep
Neutral/Phosphate 6.5g Na2HPO4 storage of surgical, Addxn:  Reduced PAS positivity
Buffered Formalin 100mL 40% CH2O post-mortem and  Prevents ppxn of formate and myelin reactivity of
(pH7.0) 900mL dist H2O research specimens pigments Weigert’s iron hematox
 Best fixative for tissues with  Reduce basophilic staining
iron pigments and elastic  Inert towards lipids
fibers
c. Formol- 90mL Sat. aq. HgCl2 3-24h Routine post-mortem  Minimum shrinkage and  Slow penetration
Corrosive/Sublimate 10mL 40% CH2O tissues hardening  Forms HgCl2 deposits
 Brightens cytoplasmic and  Not for frozen sections
metachromatic stains  Inhibits determination of
 Structures and blood well- extent of tissue
preserved decalcification
 No washing out
 Fix lipids

d. Alcoholic 80mL 95% EtOH sat’d 6 hours Immunoperoxidase  Faster fix  Gross hardening of tissues
Formalin/Gendre’s with picrate studies and EM  Fix and dehydrant at the same and partial lysis of RBCs
Fixatve 15mL 40% CH2O time  Poor preservation of iron
5mL GlHAc  Preserve glycogen pigments
 Fix sputum as it coagulates
mucus
e. Glutaraldehyde Made of 2 CH2O 2.5% - 2-4hrs 2.5% - small tissue  Preserves plasma proteins  Slow penetration Specimen containers be
residues, linked by 3 C 4% - 6-24hrs fragments and needle  Less tissue shrinkage  Makes tissue brittle refrigerated during fixation
chains. biopsies  Preserve cell structures  Reduce PAS positivity and Change solutions
4% - larger tissues  Not an irritant mucin reactivity (immerse
in conc. GHAc and aniline
oil)
FIXATIVE FORMULA/DESCRPXN FIX TIME INDICATION ADVANTAGE DISADVANTAGE PRECAUTIONS
METALLIC FIXATIVES
Mercuric Chloride Most common 24h Widely used as a  Penetrates and hardens tissue  Marked shrinkage (add Formation of black deposits
metallic fixatve secondary fixative rapidly acid) (treat with sat. iodine in 96%
Routine fixative for  Fine nuclear detail  Incomplete fixation alcohol – DE ZENKERIZATION)
5g HgCl2 preservation of cell  Pppts all proteins  Considerable RBC lysis and
2.5g KCrO2 detail in Tissue  Excellent trichrome staining removes much iron from
100mL Dist. H2O photography  Briliiant metachromatic hemosiderin
For renal, fibrin, staining  Reduces glycogen demo
connective, and  Corrosive to metals
muscle tissues
a. Zenker’s Fluid Mercuric chloride 12-24h All kinds of tissues  Rapid and even fix  Poor penetration
stock in GHAc (to Recommended for  Doesn’t disintegrate  Requires overnight
prevent turbidity and small pieces of liver,  Brilliant staining of nuclear washing
dark ppt) spleen, connective and CT fibers
tissue fibers and  May act as mordant
nuclei
b. Zenker-Formol/Helly’s 5g HgCl2 12-24h Microanatomic  Penetrates well  Same with zenker Produce brown pigments if
Solution 2.5g KCrO2 Pituitary gland, BM,  Better nuclear fix and stain exposed in for more than 24h
100mL dist. H2O blood containing than zenkers (treat with sat. alc. Picrate or
5mL 40% CH2O, added organs  Preserves cytoplasmic NaOH)
before use granules
c. Heidenhain’s Susa 45g HgCl2 3-12h Tumor biopsies (esp.  Penetrates and fix rapidly and  Prolonged fix cause Tissue must be transferred
Solution 5g NaCl skin) evenly shrink,hard and bleach directly to high-grade alc to
20g TCA Excellent cyto fix  Minimum shrinkage and  Poor RBC preserv avoid undue swelling
40mL GHAc hardening  Dissolve some cytoplmc
200mL CH2O  Easier sectioning of large granules
800mL dist. H2O blocks of fibrous CT  HgCl2 deposits
 Transferred directly to alcohol  Cannot use Weigert’s
method for elastic fiber
d. B-5 6g HgCl2 1 ½-2h BM biopsies  Rapid fix  Hardens if overfixed
1.25g NaAc  BM  Two solutions used,
90mL dist. H2O mixture is unstable
1mL/10mL B5 40%  Ppt on standing
CH2O prior to use  HgCl2 pigment
FIXATIVE FORMULA/DESCRPXN FIX TIME INDICATION ADVANTAGE DISADVANTAGE PRECAUTIONS
CHROMATE FIXATIVES
Chromic Acid 1-2% aq solution  Ppt all proteins  Strong oxidizer (Requires
 Adeq preserves carbs strong reducer)
a. KCrO2 3% aq solution  Fix, not ppt, cytoplasmic  Destroys mitochondria at
structures acidic pH
 Preserve lipid
 pH4.5-5.2: Preserve
mitochondria
 acidified pH: preserves
cytoplasm, chromatin bodies
and chromosomes
b. Regard’s/Muller’s 80mL 3% KCrO2 12-48h  Demo chromatin,  Darkens and deteriorate in
Fluid 20mL 40% CH2O, mitochondria, mitotic figures, acidity and prolonged
added before use golgi, RBC and colloid  Slow penetration
containing tissues  Produce sub-oxide ppt
(wash thoroughly)
 Lipid inert and reduced
PAS rxn
c. Orth’s Fluid 100mL 2.5% KCrO2 36-72h Study of early  Preserve myelin better than  Same as Regard’s
10mL 40% CH2O, degenerative formalin
added before use processes and tissue
necrosis
Demonstrate
ricketssiae and other
bacteria
LEAD FIXATIVE 4% aqueous solution  Fix CT mucin  Forms Lead carbonate
basic lead acetate  For Acid MPS (add acetate)

PICRIC ACID FIXATIVES 1% aq solution  Excellent for glycogen demo  RBC hemolysis and loss of Dyes yellow (treat with
 Penetrates rapidly iron another acid dye or lithium
 Brilliant staining w trichrome  Not for frozen sxns carbonate or place in
 Ppt all proteins  Hardens 70%EtOH then 5% Na
 Stable  Excessive staining thiosulfate and wash)
 Explosive when dry
a. Bouin’s solution 75mL sat picrate 6-24h For embryos and  Minimal distortion of micro  Poor penetration
25mL 50% CH2O pituitary biopsy anatomical structures  No for kidney, lipid and
5mL GHAc  Preserve soft and delicate mucus
structures  Destroy cytoplasmic
 Yellow stain for fragmentary structures
biopsy  Produce hemolysis and
 No washing out iron
b. Brasil’s alcoholic 2040mL 37% CH2O Excellent for glycogen  Less messy than bouins 
Picroformol 80g picrate
6000mL EtOH
65g TCA
FIXATIVE FORMULA/DESCRPXN FIX TIME INDICATION ADVANTAGE DISADVANTAGE PRECAUTIONS
GLACIAL ACETIC ACID Normally used in Study of nuclear  Fix and ppt nucleoproteins  Not for cytoplasmic fix
conjunction with other components  Ppt chromatin and
fixatives to form a chromosomes
compound solution.

Solidifies at 17C
ALCOHOL FIXATIVES Denatures and ppts Abs – fix and preserve  For small tissue fragments  Lower conc cause
proteins by destroying glycogen, pigments,  Both fix and dehydrant hemolysis
hydrogen and other blood, tissue films and  Preserves nuclear stains  Dissolves lipids
bonds smears  Considerable shrinkage
It is used in 80% - photographic
high=grades since less work
concentrated
solutions will produce
lysis of cells
a. Methanol 100% solution Excellent for fixing dry  Both fix and dehydrant  Slow penetration
and wet smears, blood  Considerable hardening
smears and B< tissues

b. Isopropanol 95% solution Used for fixing touch  

preparations
c. Ethanol 70-100% solution 18 – 24h Simple fixative. But  Preserve but not fix glycogen  Polarization of glycogen
incorporated as  Fix pigments, blood, tissue granules
compound for better films and smear  Considerable hardening
results  Preserves nucleoprotein and and shrinkage
NA  Strong reducer

d. Carnoy’s Fluid 60mL Abs OH 1-3h For fixing  Rapid fix for urgent biopsy  Hemolysis
30mL chloroform chromosomes, lymph  Tissue exposed 1hr can be  Considerable shrinkage
10mL GHAc glands and urgent directly transferred to abs and hardening
biopsies alcohol  Slow penetration
 Fix brain tissue – rabies  Dissolves lipids
diagnosis
 Nuclear preservation,
staining, differentiation
 Preserves Nissl and
cytoplasmic granules
 Glycogen fix
e. Newcomer’s Fluid 60mL Isopropanol 12-18h @ 3C Fix MPS and nuclear  Both nuclear and 
30mL Propionate proteins histochemical fix
10mL Petroleum ether
10mL acetone
10mL dioxane
FIXATIVE FORMULA/DESCRPXN FIX TIME INDICATION ADVANTAGE DISADVANTAGE PRECAUTIONS
OMSIUM TETROXIDE/ Pale yellow poweder Neurological studies  Fix lipids permanently  Expensive Keep in dark colored
OSMIC ACID/OSMATE that dissolves in water Ultrathin sectioning  Cytoplasmic structures  Penetrates poorly container to prevent
forming a strong  Myelin and peripheral nerves  Reduced by exposure to photoreduction
oxidant. Fiation causes  Brilliant nuclear stain diff agents Keep in fridge to prevent
complete (safranin)  Irritant deterioration
denaturation of  Ppt gel and proteins  Inhibits hematox Black deposits form (treat
protein.  Eliminates washing out  Volatile with sat aq HgCl2), may be
dissolved in water
a. Flemming’s Solution Most common 24-48h Nuclear preparation  Excellent for nuclear  Poor penetration
chrome-osmium structures  Deteriorate rapidly
acetate fix  Permanent fat fix  Depress staining of
15mL 1% aq chromate  <10 x volume reqd for fix hamtox
4mL 2% aq osmate  Form artefact pigments
1mL GHAc (wash for 24 hrs)
b. Flemming’s solution 24-48h For cytoplasmic  Same as flemmings  Same as flemmings
without acetate structures, particulary
mitochondria
TRICHLOROACETIC ACID Incorporated into  Ppt proteins  Poor penetration
(TCA) compound fixatives  Counteract shrinkage by other
fixatives
 Fix and weak decalcifying
agent
 Softening effect, facilitate pre
for dense fibrous tissue
ACETONE Used at ice cold Study of water  Fix brain tissue  Shrinkage and distortion
temperature diffusible enzymes  Dissolve fat
Diagnosis of rabies  Rapid evaporation
Solvent for metallic
salts to be used in
freeze sub techniques
HEAT FIXATION Involves thermal For frozen tissue  Preserves nuclear and  Considerable shrinkage
coagulation of tissue sections and cytoplasmic detail and distortion
protein for rapid preparation of  Hemolysis
diagnosis, bacteriological smears  Dissolve starch and
glycogen
DECALCIFICATION – calcium or lime salts are
removed from tissues following gixation and
before impregnation to ensure and facilitate
normal cutting of section to prevent
obsctruction of microanatomic detail
In hematoxylin staining, microcalcifications
appear as dark purple granular masses with
lighter purple halos
Grating sensation encountered can be
prevented by placing the block face down on a
pad saturated with 10% HCl for 1 hour
Factors Influencing Decalcification
Rate of Decalcification
 Structure
o Increased size and consistency
will require longer period for
complete decal
o Ideal time Is 24-48h
 Temperature
o Hastens decalcification
o 37C: impaired nuclear staining
of Van Gieson’s for collagen
fibers
o 55C: complete digestion within
24-48hrs.
o 18-30C: Optimum decal
 Volume of solution
o 20:1 fl to tissue volume
recommended
Measuring Extent of Decalcification
 Physical or Mechanical Test
o Done by touching or bending
tissue w fingers and determine
consistency
o Diminished consistency w softer Tissue softeners
feels indicate delcalcified tissue  For unduly hard tissues
o Vague and inaccurate  Selected portions left in fluid for 12-24h
o Alternately done mechanically and dehydrated
by pricking with a fine needle or
 Can also be done by washing out and
proble although this produces
artefacts or false positive immersion in 4% aq phenol for 1-3d
microfractures of fine without producing great distortion
trabeculae  Examples
 X-Ray or Radiological Method o Molliflex: specimens will appear
o Very expensive but the most swollen and soapy
ideal, most sensitive and most o 2% HCl
reliable
o 1% HCl in 70% Alcohol
o Appears opaque in a plate
o Bone placed on polyethylene
sheet on top of Xray film and
exposed for 1min @ 30kV
 Chemical Method/CaOx test
o Simple, reliable and convenient
for routine purposes
o Precipitation of insoluble CaOH
or CaOx
Post decalcification
 Done by immersing decalcified bone in
either saturated lithium carbonate or 5-
10% aq NaH2CO3
 Rinsed with tap water for 30 minutes
for small samples and 1-4hr for larger
samples
 Frozen sections thoroughly washed in
water or stored in formol saline with
15% sucrose or PBS with 15-20%
sucrose at 4C before freezing
 Tissue decalcified with EDTA: do not
place directly into 70% alcohol, cause
precipitation
DECALCIFYING AGENT
ACID DECALCIFIERS
Nitric Acid
a. 10% aq Nitric Acid
b. Formol-Nitric Acid
c. Perenyi’s Fluid
d. Phloroglucin-Nitric Acid
Hydrochlroic Acid
a. Von Ebner’s Fluid
b. Formic acid
C. Formic Acid-sodium
citrate solution
Trichloroacetic acid
Sulfurous acid
Chromic acid (Flemming’s)
Citric Acid-citrate buffer
solution (pH4.5)
DESCRIPTION/FORMULA
Routine large amounts bony
tissues
Most common and fastest
decalcifier (routine)
Used as simple or combined
5-10%
10mL conc nitric acid
100mL dist H2O
10mL conc nitric acid
5mL 40% formalin
85mL dist. H2O
40mL 10% nitric acid
30mL 0.5% Chromic acid
30mL abs EtOH
10mL conc. Nitric acid
1g phloroglucin
100mL 10% nitric acid
50mL 36% sat aq NaCl soln
8mL conc HCl
50mL dist H2O
M10mL formate
90mL 10% formal saline
50mL 20% aq sodium citrate
50mL 45% formate
5g TCA
95mL 10% formol saline
Very weak decalcifier suitable
only for minute pieces of bone
15mL chromate
4mL 2% osmic acid
1mL GHAc
5mL 7% citrate
95mL 7.4% ammonium citrate
0.2mL 1% ZnSo4
DECALCIFYING TIME INDICATION ADVANTAGE DISADVANTAGE
Stable
 Rapid  Destroys if concentrated
 Minimal distortion  Impaired staining (treat
with formalin or alcohol)
12-24h Urgent biopsy  Rapid  Distorts if prolonged
Needle biopsy  Minimal distortion  Imparts yellow color
Large or heavily mineralized  Good nuclear staining
cortical bone  Easily removed by 70% OH
1-3d Urgent biopsy  Rapid  Yellow color (neutralize
 Less destruction than 10% nitrate with 5% NaSO4 and was for
12h)
2-7d Routine  Decalcifier and softener  Slow for dense bones
 Good staining  Ppt fmxn in addition of NH3
 No maceration reqd
12-24h Urgent works Most rapid decalcifying agent  Poor nuclear staining
 Distorts if prolonged
 Yellow color, same
neutralization indicated
 Cannot determine
complete decalcification
Surface decalcification of  1% soln with 70% alcohol: good Inferior to nitric acid as a
blocks nuclear stain decalcifier
Cytologic staining  Moderately rapid and does not Extent of decalcification
For teeth and small bone require washing cannot be measured
pieces
2-7d Routine for post-mortem  Fix and decal  Slow
research tissues  Safer than nitrate and hcl  Neutralize with 5% sodium
Primary decalcifier  Routine surgical specimens for sulfate
immunohistochemical staining
3-14d Autopsy materials, BM,  Better nuclear staining than nitrate Same as formate
cartilage for research
4-8d  Good nuclear staining  Weak
 No washing out; excess removed  Slow-acting
by several changes in 90% alc.
For minute bone pieces  Fix and decal  Inhibits hematox
 Insoluble pigments formed
 Cannot measure extent of
decal
6d  Excellent nuclear and cytoplasmic Too slow for routine purposes
staining
 No distortion
DECALCIFYING AGENT DESCRIPTION/FORMULA DECALCIFYING TIME INDICATION ADVANTAGE DISADVANTAGE
Chelating agent (EDTA) Substances which combine with Small specimens: 1- Detailed microscopic  Excellent staining  Very slow
calcium ions and other salts forming 3wks studies (EM)  Minimal distortion and  Requires change every 3 days
weakly dissociated complexes Dense cortical bone: Immunohistochemical artefacts  Does not bind at pH3
facilitating removal of calcium salt. 6-8wks staining  Extent of decalcification can be  Faster binding at pH7-7.4 and
measured optimal at pH8 and above
5.5g EDTA disodium salt  Slight hardening
90mL dist. H2O  Inactivates ALP (restored by
10mL 40% formalin adding MgCl20

Ion Exchange Resin Hastens decalcification by removing  Cellular detail preserved  Extent of decalcification cannot
Ca ions from formate-containing  Hastened decalcification be measured by chemical
decalcifiers, increasing solubility  No washing methods
from tissue  Excellent staining  Very slow
 Minimal distortion and  Slight hardening
Layer of exchange resin is spread artefacts
over the bottom and specimen  Extent of decalcification can be
placed on top measured with xray method
Decalcifier is added, 20-30times the
volume
Electrophoresis Positively charged calcium ions are Small bone fragments   Prolonged decal: prevent
attracted to a negative electrode hydrolysis, maceration and
and subsequently removed from the destruction
decalcifying solution  May be incomplete

Shortened decalcification due to
heat and electrolytic reaction

Same principle with chelation

except for application of heat

100mL 88% formate

80mL conc HCl
1000mL dist. H2O
DEHYDRATION
 Removal of fixative and water from the tissue
 Replacement with dehydrant prior to impregnation
 Removal of intercellular and extracellular water from the tissue following fixation and prior tom impregnation
 Usually starts with 70% EtOH progressing through 95% and finally 100%.
o Delicate tissues (Embryonic): Start with 30%
 Keep time brief
 General rule: Volume at least 10 times the volume of the tissue
 Ideal dehydrant:
o Rapid without producing considerable shrinkage and distortion
o Does not evaporate fast
o Dehydrates even fatty tissues
o Does not harden excessively
o Should not remove stains
o Not toxic to the body and not a fire hazard
For ALCOHOL

 Tissue passed through progressively increasing concentration.

 Transfer of formalin-fixed tissue to high-grade alcohol directly: Great shrinkage and hardening
 Concentrated alcohol: Harden only surface resulting to unequal impregnation and poor cutting later
 Prolonged storage in lower concentration: Maceration of tissue
 37C temp hastens dehydration
 Confirm complete dehydration: Add anhydrous copper sulfate (blue discoloration indicates full saturation with water)
 Order:
Susa/Carnoy’s/Formol- 10% Formol-Saline Bouin’s Zenker/Helly’s Flemming’s
sublimate
Running Water 1-12 1-12
30% Alcohol 1-6 ½-3
50% Alcohol 1-6 ½-3
70% Alcohol 13-5 13-12 1-6 ½-3
90% Alcohol 1-6 13-5 13-12 1-6 1-3
Absolute Alcohol 1-6 13-5 13-12 1-6 1-3
Absolute Alcohol 1-6 13-5 13-12 1-6 1-3
Absolute Alcohol 1-6 13-5 13-12 1-6 1-3

Additives to Dehydrants
4% Phenol to 95% EtOH: acts as softener for hard tissues (NAIL, TENDON, DENSE FIBROUS TISSUE)
DEHYDRATING AGENT
ALCOHOL
1. Ethyl Alcohol (Ethanol)
2. Methyl Alcohol
(Methanol)
3. Butyl Alcohol (Butanol)
ACETONE
DIOXANE (Diethylene
Dioxide)
Cellosolve (Ethylene glycol
monoethyl ether)
Triethyl Phosphate
Tetrahydrofuran (THF)
DEFINITION/FORMULA
Clear, colorless
Considered the best
dehydrating agent
Clear, colorless
Clear, colorless
Considered the best
dehydrating agent
A very good dehydrating
agent but not used in the lab
as a victim of tradition
DHYDRATION TIME INDICATION
  For blood and tissue
films
 smear preparation
  plant and animal micro-
techniques
 tissues that does not
require rapid processing
 ½-2h  For small pieces of tissue
 Graupner’s method: 4hrs,  Both dehydrant and
3 changes Pure dioxane clearing
(1,1,2) and 3hrs, 3
changes Paraffin
(0.15,0.45,2) (T:7hrs)
 Weiseberger’s: tissue
wrapped in gauze,
suspended in dioxane and
anhydrous CaO (3-24h)
Dehydrate sections and
smears following certain
stains
Demixing, clearing, and
dehydrating paraffin
sections before and after
staining
ADVANTAGE DISADVANTAGE
 Fast acting  Flammable
 Not poisonous
 Not expensive
  Toxic
 Less shrinkage and  Slow
hardening compared to
EtOH
 Cheap  Poor penetration
 Rapid  Highly flammable
 Miscible with H2O and most  Brittleness when prolonged
organic solvents  Extremely volatile
 Readily miscible in water,  Ribbon poorly
melted paraffin, alcohol,  Toxic vapors
and xylol  Produce explsive peroxides
 Less shrinkage compd to alc  Chromate requires wash with
 Prolonged exposure does tap water prior to dioxane
not affect consistency or treatment
staining
 Dehydrates rapidly  Combustible @ 110-120F
 Can be transferred directly  Toxic
to cellosolve and stored for
months without distortion
 Transferred directly to TEP 
 Removes water very readily
 Soluble in alcohol, water,
ether, benzene, chlor,
acetone, and xylene
 Minimum shrinkage
 Both dehydrant and clearant  Toxic if ingested or inhaled
(miscible in water and  Skin and eye irritant
paraffin)
 Soluble same as TEP
 Less shrinkage
 Easier cutting with few
artefacts
 Does not dissolve aniline
dyes
CLEARING
 De-alcoholization
 Alcohol or dehydrating agent is removed
 Alcohol is replaced with a substance that will dissolve wax which the tissue is to be impregnated with
 Complete replacement of dehydrant: Translucent appearance
 Good clearing agent:
o Miscible with dehydrating agent, embedding media and mounting media
o It should not distort and harden tissues nor should it dissolve aniline dyes later
o It should not evaporate quickly
o Makes tissues transparent
 Clearant with low boiling point: Rapidly replaced by melted paraffin ( does not apply to chloroform when compared with xylene)
 Prolonged exposure: Gives brittleness and difficulty to cut
 CLEARING AGENT DESCRIPTION
Xylene Colorless clearing agent and
used for clearing embedding
and mounting media
CLEARING TIME INDICATION ADVANTAGE DISADVANTAGE
½-1hr Routine processing less than  Most rapid, can clear within 15-30min  Inflammable
24h  Gives transparency  Considerable hardening and
Tissue block <5mm thick  Miscible w alc and paraffin shrinkage and brittleness
Urgent biopsies  Does not extract out aniline dyes  Milky when tissue is
 Evaporates quickly in paraffin oven incompletely dehydrated

Toluene 1-2h Substitute to benzene or xylene  Miscible w alc and paraffin  Slower than xylene and benzene
in embedding and mounting  No excessive hardness and brittleness after  Acidifies (in partially filled vessel:
Routine exposure 24h WHEN THERE IS AIR)
 Noncarcinogenic  Toxic fumes
 Expensive
Benzene 15-60min Preferred as clearant in the  Penetrates and clears tissues rapidly  Inflammable
embedding process  Volatilizes in paraffin oven (easy elimination)  Considerable shrinkage if left too
Urgent biopsies  Miscible with Abs alcohol long
 Does not make tissue hard & brittle  Toxic: BM damage – aplastic
 Minimum shrinkage anemia
 Makes tissue transparent
Chloroform 6-24h Routine work for larger  Little brittleness  Slow clearing than xylene in
specimens and thick blocks  Can process thicker blocks up to 1cm embedding process
 Miscible with Abs alcohol  Tissues do not become
 For tough tissues and large specimens translucent
 Not inflammable  Hepatotoxic
 Not volatile in paraffin oven
 Tissues tend to float in
chloroform (Wrap with gauze)
 Evaporates quickly in water bath
Cedarwood oil 2-3d, requires two Clear paraffin and celloidin in  Very penetrating  Extremely slow
changes embedding process  Miscible with 96% alcohol  Hard to eliminate
For CNS tissues  Minimial shrinkage and hardening  Quality not uniform
Cytological Studies (Smooth  Can be left indefinitely  Milky upon prolonged storage,
muscles and skin)  Does not dissolve aniline dyes requires filtration before use
 Makes tissue transparent  Expensive
 Improves cutting
Aniline Oil Recommended for clearing  Clears 70% alcohol without excessive shrinkage 
embryos, insects, and very and hardening
delicate specimens
Clove oil  Minimum shrinkage  Tendency to become adultered
 Wax impregnation after
becomes slow and difficult
 Brittleness
 Aniline dyes removed
 Celloidin dissolved
Carbon tetrachloride Properties similar to  Used in clearing in embedding  Same w chloroform
chloroforom but relatively
cheaper
Methyl benzoate and Double embedding techniques   Slow-acting
Methy salicylate
IMPREGNATION/INFILTRATION A. Manual Processing ii.Place in molten wax, in vacuum
 Clearing agent is completely removed from the tissue  Four changes of wax, 15mins intervals chamber and close airtight
 Replaced by a medium that will fill all the tissue  It is then immersed in another fresh iii. Exhaust air untile there is
cavities solution for 3 hours to ensure complete negative pressure 400-500mm
 Gives firm consistency allowing easier handling and embedding iv. Leave for 15 minutes then slowly
cutting readmit air until normal atp
B. Automatic Processing v. Place tissue in fresh wax
I. PARAFFIN WAX IMPREGNATION  Makes use of processing machine vi. Repeat step iii-iv
 Simplest, most common, and best media for routine  2-3 changex of wax are required to remove vii. Place in fresh wax
processing the clearing agent and properly impregnate viii. Repeat step iii and leave for 30-45
 Advantages: the specimen minutes
o Serial sections cut with ease and without o Made possible due to constant ix. Bring to normal atp and embed
undue distortion tissue agitation accelerating and
o Rapid, allows sections to be prepared w/in improving penetration Factors Influencing Paraffin Wax Impregnation
24h o Elliiot Bench-Type Processor :  Processing used: vacuum impregnation
o Tissues may be stored in paraffin for  Frequency of fluid replacement depends on gives fastest results
indefinite period without considerable number and size of tissues processed  Total impregnation time: depends on
destruction o Presence of odor of clearing nature and size of tissue and clearing agent
o Permits good staining agent: Change paraffin wax
 Disadvantages:  Wax bath: 3 degrees above melting point of
o Brittle when overheated the wax
o Prolonged IMPREGNATION results to
excessive shrinkage and hardening C. Vacuum Embedding Thickness Benzene/Toluene Chloroform/Cedarwood
o Inadequate IMPREGNATION promotes  Wax impregnation under negative Period # of Period in # of
retention of clearing agent atmospheric pressure inside an embedding in changes wax Changes in
 Soft and shrunken oven molten in wax wax
 Crumbling when sectioned o This hastens removal of air wax
 Breaks up in floating out bubbles (such as in lungs) and <3mm 1hr 1 1½h 2
o Tissues difficult to infiltrate requiring clearing agent from the tissue
3-5mm 1-5hrs 2 2-5hrs 3
prolonged immersion, however is not block
5mm- 3-6hrs 2 4-8hrs 3
advisable  Speeds up wax impregnation
1cm
o Not recommended for fatty tissues  For urgent biopsies
 For delicate tissues: >1cm 6- 3 8-12hrs 4
 Processing: o Lung 12hrs
o After clearing, Submerge in 2 or more o Brain
changes of melted wax in paraffin oven or o Connective tissues
incubator @ 55-60C. 56C wax in for routin o Decalcified bones
o Hard tissues require wax with higher o Eyes
melting point. o Spleen
o CNS
o 3 ways this is performed  This reduces impregnation by 25-75% of the
 Manual original time
 Automatic  Maintained in a thermostatically controlled
 Vacuum water-jacked maintained at 2-4C above
melting point of the wax
 Procedure after fixation and dehydration:
i. Clear, 2 changes in xylene, 1hr
each
Precautions in Paraffin Wax Impregnation 3. Ester Wax
 Mostly PEGs with MPs of 38-42C or 45-56C 2. Dry Celloidin Method – for processing whole eye
Prolonged Shrinkage and Do not leave for  Carbowax Principle and procedure: Same as wet celloidin, but
exposure to Hardening longer period o Most commonly used instead uses Gilson’s mixture (equal parts of
media o 18 or more C atoms chloroform and cedarwood oil) for storage
o Solid at RT
Overheating Srhinkage and Maintain 2-5C
o Soluble and miscible w water 3. Nitrocellulose Method – uses low viscosity
above 60C hardening and above melting
o Tissues are fixed, washed out and transferred nitrocellulose (LVN)
Complete point
directly to carbowax  Soluble in equal concentration of ether and
destruction of
o Hygroscopic, it is easily dissolved in water alcohol
lymphoid
 Low viscosity, can be used in high conc and
Wax should be II. CELLOIDIN IMPREGNATION still penetrate rapidly
pure  Purified nitrocellulose soluble in many solvents  Tendency to crack: add plasticizer
 For specimens with large hollow cavities that tend to
collapse III. GELATIN IMPREGNATION
 For hard and dense tissues (bones and teeth)  Used only when dehydration is avoided and when it is
 Large sections of whole embryo to be subjected to histochemical and ezyme studes
Filtered before Filter with coarse  Thin:2%; Medium: 4%; Thick: 8%  Used as embedding media for delicate specimens and
use in a wax filter paper: frozen sections
oven at 2C Green’s # 904 Advantages:  Water soluble; no dehydration and clearing
higher than 1. Permits cutting of sections thicker than in paraffin; for  Tissues not be more than 2-3mm thick
melting point neurological tissues  1% Phenol prevents growth of molds
2. Rubbery consistency: allows cutting of very hard or  Excess: Removed by floating out
variable consistency tissues w/o undue distortion  Volume: At least 25 times the tissue volume
Substitutes for Paraffin 3. Does not require heat 10% gelatin with 1% 24h
phenol
1. Paraplast Disadvantages: 20% gelatin with 1% 12h
 Mixture of highly purified paraffin and synthetic plastic 1. Very slow phenol
polymers 2. Thin sections difficult to cut and serials are difficult to 20% gelatin with 1% Until impregnation and
 MP: 56-57C prep phenol and cooled in ref embedding are
 More elastic resilient than paraffin permitting cutting of 3. Inflammable vapour completed
bones and brain blocks easily 4. Difficult to obtain photomicrographs 10% formalin 12-24h (Hardening)
 No deposit left in slide after staining 5. Very volatile
 Does not tend to crack like other substitutes
 Embeddol – 56-58C MP, less brittle and less Methods for Celloidin Impregnation
compressible than paraplast 1. Wet Celloidin Method - For bones, teeth, large brain
sections, whole organs
 Bioloid – semisynthetic, for eyes
Alcohol (equal parts) 12-24h
 Tissue Mat – contains rubber
Medium celloidin 5-7d
2. Ester Wax Thick celloidin 3-5d
 Lower melting point, 46-48C Embedding medium
 Harder than paraffin w thick celloidin
 Soluble in 95% EtOH but not in water and other Keep in jar or
clearants (can be used for impregnation w/o prior dessicator
clearing) Top is removed
Check by touching
Store in 70-80% alc Until use
EMBEDDING/CASTING/BLOCKING
 Impregnated tissue is placed into a mold w medium
and allowed to solidify
 Arranged at the bottom of the mold and immersed in
melted paraffin at 5-10C above its MP and cooled
rapidly in -5C or immersed in cold water to solidify
 Orientation: Process by which a tissue is arranged in
precise positions
Types of Blocking-out Molds used:
1. Leuckhart’s Embedding Mold
 2 L-shaped strips of heavy brass or metal arranged
on a flat metal plane
 Adjusts size of mold to specimen
 Even blocks with parallel sides
 Routine use
2. Compound Embedding Unit
 Series of interlocking plates resting on a flat metal
base
 Forms several compartments
 More specimens embedded at a time
3. Plastic Embedding Rings and Base Mold
 Stainless steel base fitted with plastic embedded
ring (block holder later)
OTHER EMBEDDING METHODS (PREVIOUSLY USED) Sum up:
1. Celloidin or Nitrocellulose Method – tissues embedded in  Use acrylic plastic sections for high resolution light
shallow tins of enamel pans covered by sheets of weighted microscopy
glass. Use of celloidin was discouraged due to special  Stain with the plastic in situ
requirements 1. Alternative of mma permits plastic to be dissolved
2. Double-Embedding Method – tissues first infiltrated with
celloidin and subsequently embedded in paraffin mass.
Used to facilitate cutting of large blocks of dense firm
tissues
IV. PLASTIC (RESIN) EMBEDDING
 Superior results for light microscopy
 High resolution for thinner sections
1. Epoxy
 Balanced mixture of epoxy plastic, catalysts and
accelerators
 Three types:
 Bisphenol A (Araldite) – slow infiltration
 Glycerol (Epon) – low viscosity
 Cyclohexene dioxide (Spurr) – low viscosity
 Disadvantages:
 Hydrophobic
 Antigenicity compromising immunihistochm
staining
 Sensitizer

4. Disposable Embedding Molds 2. Polyester

a. Peel-Away
 Thin plastic mold which is peeled off one
at a time as soon as wax has solidified
 Can be placed directly in chuck
b. Plastic Ice Tray
 Used in ordinary ref
 For busy routine labs
 Compartment may be utilized for 1 block,
removed by bending the plastic tray
c. Paper Boats
 For embedding celloidin
 For EM, but were suspended by epoxides superior to it
3. Acrylic
 Made up of esters of acrylic or methacrylic acid
 For light microscopy
 Polyglycol methacrylate (GMA)
 Extremely hydrophilic, allowing staining
 Tough when dehydrated
 Methyl methacrylate (MMA)
 Hardness ideal for undecalcified bone
 Benzoyl peroxide added as catalyst to for phenyl
radicals forming active sites to be polymerized by
acrylics
 Methyl methacrylate (MMA)
 Hardness ideal for undecalcified bone
 Stored in dark bottles to preven premature
polymerization due to radicals formed by heat or light
MICROTOMY  Different from a rocking microtome as E. ULTRATHIN MICROTOME
 Processed tissue is trimmed and cut into uniformly o sections are cut in a perfectly flat plane, allowing  For electron microscopy
thin slices or sections excellent serials  Sections at 0.5 microns
 To facilitatie studies under the microscope o heavier and more stable  Knife: selected fragments of broken plate glass
 Sections are cut at a predetermined thickness by o more complex in design and construction  Specimen: small, fixed in osmic acid and embedded in
sliding the block into a knife fixed to the machine  Relatively dangerous as the blade is placed up plastic
 Essential parts:
o Block Holder – holds the tissue block in position C. SLIDING MICROTOME MICROTOME KNIVES
o Knife Carrier and Knife – cutting of tissue sections  Developed by Adams in 1789
o Pawl, Ratchet Feed Wheel and Adjustment  Types: Knife Length Characteristic Microtome Media
Screws – Line up tissue block in proper position o Base-Sledge microtome Plane- 25mm 1 side flat Sliding Celloidin
with the knife, adjusting proper thickness of the  Knife can be set at an angle for cutting Concave and another microtome
tissue for successive sections celloidin since it has 2 movable pillars biconcave
 Principle: holding adjustable knife clamps Biconcave 120mm 2 sides Rotary Paraffin
concave microtome
o A spring-balanced teeth or pawl is brought into  Block holder, on a metal base, can be
Plane- 100mm 2 sides Base- Frozen
contact with, and turns a ratchet feed wheel moved backwards and forwards under a Wedge straight sledge/Sliding sections
(connected to a micrometer screw, which is in stable knife Knife microtome or
turn rotated, moving the tissue block at a  Favored when hard tissue or large blocks extremely
predetermined distance towards the knife cutting are sectioned hard and
sections at uniform thickness  Suited to section all forms of media; cuts in tough
 5 kinds of Microtome: a perfectly flat plane specimens
o Rocking microtome o Standard Sliding microtome
o Rotary microtome  Block remains stationary while the knife is A cutting facet/bevel is found on the tapered edge of a knife,
o Sliding microtome moved backward and forward forming a cutting edge
o Freezing microtome  Mainly for celloidin tissue blocks Bevel angle
o Ultrathin microtome  Both types can be set obliquely for CELLOIDIN or  Angle formed between cutting edges
strainght for LARGE REFRACTORY PARAFFIN blocks  About 27-43 degrees
A. ROCKING MICROTOME  Kept constant by a slide-on back.
 Invented by Padlwell Trefall in 1881 D. FREEZING MICROTOME
 For cutting serial sections of large blocks of paraffin  Invented by Queckett in 1848 Good cutting edge
embedded tissues  For unembedded frozen sections  Good quality steel
 Simplest among the other microtomes  Stage for block holder: Hollow and perforated o Too soft cutting edges: likely to become dull
 Heavy base and two arms  Has a reinforced flexible pipe which carbon dioxide o Too hard edges: likely to produce nicks or jagged
o Lower arm rests on pivots passes thru which is released in intermittent bursts by edges and irregularities on the edge producing
o Upper arm carries block holder, connected a lever tears or striation
to a lever by a nylon thread  Another cooling device is present to lower temp of  Cut good sections from a paraffin block about 2-
o Cuts at about 10-12 microns knife 3microns thick without serrations
 Cambrdige rocking microtome:  Rapid diagnosis, histological demonstration of fat, o Safety razor blades: for calcified materias, paraffin
o Cut small and large blocks study of neurological structures, heat-sensitive and frozen sections, sectioning unsatisfactorily for
o Theoretically not recommended for serial tissues sections less than 10 microns
sections as it cuts in slightly curved planes  Cryostat/Cold microtome: o Optimum angle: 15 degrees, causes maximum
o Not currently favoured by most laboratories o Refrigerated apparatus penetration and minimize distortion
due to restrictions in size of tissue block o Fresh tissue microtomy  Must be thinner than the section being cut
o Freezes tissueinto the block holder to the correct  Sharp enough to cut good sections
B. ROTARY (MINOT) MICROTOME degree of hardness
 Invented by Minot in 1885-1886 o Microtome is maintained at -5C to -30C Clearance angle
 For cutting paraffin embedded tissues o Cuts 4 microns  Angle from the knife to the cutting plane
 Adjustment screws made so the block is parallel to  About 5-10 degrees, so that the cutting facet will not
the microtome knife at all planes compress block during cutting
HONING AND STROPING  Precautions:
o Hone be long (8” x 3”)
Honing o Hone be lubricated before use
 Removal of gross nicks on the knife edge: to remove o Wash with water to remove metal particles
blemishes collected, fluid must flow rapidly
 Grinding of cutting edge on a stone: Acquisition of o Pressure during honing should be gentle
even edge and steady
 Degree of sharpness is proportional to fineness of o Strokes are usually 20-30 per direction
abrasive used depending on knife
 Makes use of a hone: A sharpening stone or grinding
surface: Stropping
o Belgium Yellow  The process whereby “burrs” formed during honing is
 Manual sharpening removed and cutting edge is polished
 For edge rendered blunt or nick  To polish and sharpen cutting edge
 Gives the best result  Paddle strop made of quality horse leather used;
o Arkansas firmly attached to a solid back to prevent sagging
 Same as Belgium yellow but with a better  Done KNIFE BACK FIT FIRST, THEN TOE TO HEEL AND
polishing effect EDGE LAST
o Fine carborundum o 40-120 double strokes
 Coarser than the two o Minot / Plane-wedged knife: turned over
 Only for badly nicked knives per end of stroke
o Plane-concave knife: concave surface only
 Maintenance of hone:  Precautions:
o Wipe with soft cloth moistened with xylene o Knife wiped clean prior to stropping and
 To remove scattered small particles of stone prior to changing from coarse to fine strop
and metal o Knife edge be oiled or greast after
o Covered with a thin film of lubricant stropping: this prevents rusting
 Mineral and Clove Oil o Knife not be allowed to rest on its sides:
 Xylene may damage cutting edge
 Liquid paraffin o Pressure: light at first stroke, leather has
 Soapy Water natural compressibility (this does the work)
and a slip may cut the strop and damage
 Done EDGE FIRST, HEEL TO TOE the cutting edge
o 10-20 strokes o Speed should be avoided: Make full strops:
o Minot / Plane-wedged knife: knife turned over to avoid injury both to strop and knife
per end of stroke o Leather strops
o Plane-concave knife: concave surface only  Usually dry, thus requires oiling
 Treated with vegetable oil
 Aluminium oxide with water can be used as abrasive  Oil applied AT THE BACK OF THE STROP,
for grinding NOT THE SURFACE
 Diamantine can be used for final polish  Used AT LEAST 24-48HRS AFTER
 Done on a plate glass TREATMENT
 Mechanical honing: vibrating frosted glass plate or a o MINERAL OIL SHOULD NEVER come in
wheel driven by an electrical motor; 30 double contact: blisters and destroys leather
strokes done (permanent blemish)
o WAX NEVER come in contact with strop
o NEVER WIPE PAPER OR CLOTH ON knife but
wipe KNIFE ON cloth or paper after xylene
wash for wax removal
Disposable blades
 Gets rid of honing and stropping
 Cheaper than conventional steel knives
 Cuts 2-4 microns thick sections with ease
 Magnetic knives used in cryostat
Glass knives
 Trimming and semi-thin sectioning of tissue blocks
 For electron microscopy
Diamond knives
 Cut any type of resin block
 For electron microscopy
 Mounted on ultrathin microtome
 Brittle and expensive, but very durable
OTHER EQUIPMENT USED FOR SECTIONING
1. Waterbath
 Temp: 10C below MP of paraffin wax
 Detergent may be added to reduce surface
tension (allows flattening)
2. Drying oven or hot plate
 For drying of sections
 Hot plate as alternative
 Splitting and cracking of sections of delicate
tissues: Set at an even lower temp
3. Forceps and squirrel hair brush
 Handling of sections during cutting
 Remove folds and creases during floating out
4. Clean Slides
 Routine: 76x25mm, 1-1.2mm thick
5. Ice Tray
6. Pencil
SECTIONING
 Tissues are cut into uniformly thin slices or
“SECTIONS”
 Facilitates studies under the microscope
 General types of tissue sections:
o Paraffin sections
 For paraffin embedded blocks cut by
rocking and rotary microtome
o Celloidin sections
 For celloidin embedded tissues cut by
sliding microtome
o Frozen sections
 For tissues fixed and frozen with CO2
or for freshe tissues frozen with
cryostat
A. PARAFFIN SECTIONS
 Trimming: Excess wax is cut off from a
solidified block
o Thin slices at a time: Prevents cracking of
block
o Trimmed until perfectly level and parallel
 After trimming, block is hardened for
cutting proper
o Faced down in ice cold water; or
o Referigerated 5-10mins.
o Cooling tissue and wax: Gives both a
similar consistency
 Final trimming:
o Setting thickness adjuster at 15mm; or
o Advancing block (using coarse feed
mechanism)
o Surface block is trimmed until it is partly
exposed
 Proceeding to cutting:
o Done until sections come out
o Cutting rhythm is maintained
o Rate depends on type of tissue, size of
block and type of microtome
o Routine: 4-6 microns
o Knife: tilted at 0-15 degrees: Allows
clearance angle
 Biconcave knives require smaller
clearance angle than wedge-shaped
knives
o Ribbons form due to slight heat
generated between block and knife
 Removed in ribbons of ten
 Floating out
o To flatten sections and prepare them for
mounting
o Folds and reases removed by stretching
gently with forceps or dissecting needle C. FROZEN SECTIONS
o Shoud not be left for a long time: Causes
undue expansion and distortion
o Done 6-10C lower than the melting point
of the wax
o Section is picked up onto a clean slide
and is immersed in water bath vertically
o Alternatives:
 Place on slide flooded with 20%
alcohol (This produces convection
currents removing creases)
 Few drops of distilled water on slide
and gently warmed on a hot plate and
drained
 Drying of Mounted Sections
o Paraffin oven: Maintained above 2-5C of
wax’s melting point
o Incubator:
 37C: Routine
 45-55C: Enzyme digestion or
localization, chemical extraction,
metallic impregnation
o Hot plate not recommended: Risk of
overheating and exposure to dust
B. CELLOIDIN SECTIONS
 Cut 10-15 microns
 Trimmed in the same manner with paraffin
blocks
o Difference: Does not require hardening
by chilling prior to cutting
 Cut with sliding microtome
 Cut by Wet Method: Avoids dehydration
and shrinkage
 Celloidin sections DO NOT COME OFF IN
RIBBONS
o Collected into 70% alcohol immediately
 For rapid diagnosis of tissue in question
 Demonstration of lipids and carbohydrates
 Immunofluorescent and
immunocytochemical staining
 Methods of preparation:
o Cold knife
o Cryostat
A. COLD KNIFE PROCEDURE
 Filter paper soaked in gum syrup placed on
stage and bursts of CO2 are applied
 Tissue block, 3-5mm thick, is oriented and
gum syrup is applied and frozen with CO2
o Frozen just to be firm enough to be
sectioned
 It is trimmed until flat
 The finger is used to warm the surface until
tissue thaws and become visilble - - This
point is the DEW LINE
 Cut at 10 microns
 NO RIBBONS:
o Removed and ransferred to distilled
water
 Do not freeze too hard: Chips into
fragments during cutting
 Insufficient freeze: cuts thick and crumbles
  Controlled environment B. ISOPENTANE COOLED BY LIQUID NITROGEN
o Knife – 40-60C
o Tissue – 5-10C
o Environment – 0-10C
B. CRYOSTAT PROCEDURE
 Uses cryostat
 Insulated microtome maintained at -20C
 Optimum working temp: -18 - -20C
 Tissue
o Should be fresh
o Done quickly as possible: Slow freeze C. AEROSOL SPRAYS
produces ice crystal artifacts
 Commonly used freezing methods:
o Liquid nitrogen
o Isopentane cooled by liquid nitrogen REQUIREMENT OF FRESH FROZEN TISSUE
o Carbon dioxide gas
o Aerosol sprays
A. LIQUID NITROGEN
 For histochemistry
 In intra-operative procedures
 Most rapid of the commonly available
freezing agents
 Soft tissues can crack due to rapid
expansion of ice
 Overcools urgen biopsy blocks; damages
both block and blade if done at -70C. : allow
nitrogen to equilibriate to cryostat temp
 Non-fatty unfixed tissues: sectioned at -10 -
-25C
 Causes vapour phase that forms around the
tissue and acts as an insulator resulting to
uneven cooling: Freeze in freezant with high
thermal conductivity (isopentane, Freon
2.2, OCT)
 Liquid at RT
 Process:
o Isopentane in a container is
suspended in a container with
liquid nitrogen until small crystals
form (approx.. -170C). It is then
removed.
o Tissue dropped into cooled
isopentane
 Used for small pieces of tissue EXCEPT
MUSCLE
 Tissue be maintained in frozen solid state
during cutting to prevent damage and
distortion by the knife
 Tissue be sufficiently cold and hard to
prevent compression and displacement
 Not be colder than the knife: Creates
resistance to cutting; becomes brittle and
breaks in cutting
REQUIREMENTS OF MICROTOME KNIFE
 Chilled and maintained at low temp:
Prevent melting
 Lower temp required for specimens in a soft
matrix e.g.:
o Fat
o Mucin
o Hard dense structures
MOUNTING OF TISSUE BLOCK
 Mounting media used:
o Water-soluble glycol
o Water-soluble resin
o Has different temp ranges:
-5 to -15C Brain, lymph nodes, liver,
spleen, uterine curetting, soft
cell tumors
-15 to -25C Non-fatty breast, ovary,
prostate, tongue, GI tract
-35 Fatty breast, omental tissue
 Tissue should be 2-4mm: minimizes knife
hitting metal block holder
 Small tissue fragments (curettings, brain
biopsies)
o placed on thick OCT base and
covered with additional matrix of
OCT and frozen by liquid nitrogen
MOUNTING OF CRYOSTAT SECTIONS
 1 edge of glass slide rested on knife surface,
other end lowered gently until 0.5-1mm
from face of knife
 Do not press section down: cause frost
mark to remain
 Cryostat CUT ONLY INDIVIDUAL SECTIONS
SPECIAL PROCESSING TECHNIQUES  Alternative:
 Freeze-drying o Rapid freezing to -160C in
 Freeze substitution isopentane supercooled liquid
 Principle: nitrogen
o Freezing causes instant cessation of o 8-10 microns
cellular activity preventing chemical
alteration of constituents and FAULTS DURING TISSUE PROCESSING
displacement of cellular tissue FAULT REASON REMEDY
components Brittle or fixation Prolonged Softened by
hard tissue dehydration soaking in
A. FREEZE-DRYING clearing WATER WITH
 Rapid freezing of tissues to -160C infiltration DETERGENT,
Overheated PHENOL, OR
 Dessication: Transfer frozen block in MOLLIFLEX
paraffin
vacuum at a higher temperature without Drying of tissue
any chemical fixative before fixation
 Restricted to specialized/research labs Clearing Incomplete dehydration Re-dehydrate
 Tissue plunged into isopentane or agent with absolute
propane-ispoentane chilled to -160 - -180C becomes alcohol
milky
with liquid nitrogen: Solidifies tissue 2-3s, Smell of Insufficient impregnation Trim block
preventing crystals clearing Melt wax
 Tissue transferred into vacuum drying agent Repeat
apparatus (water is sublimated and impregnation
Opaque Insufficient clearing Repeat
dehydrated); complete dessication in 24-
tissue clearing
48hrs Tissue Insufficient dehydration Repeat whole
 Time-consuming and expensive shrinks procedure
 Freeze-dried materials are difficult to when
section trimmed
Soft when Incomplete fixation Repeat whole
 Advantages
trimmed procedure
o Produces minimum shrinkage Air holes Incomplete impregnation Repeat
o Tissue processed as fresh impregnation
o Less displacement of tissue and cell Wax Contaminated wax Re embed in
consituents appears Block not cooled rapidly freshly filtered
crystalline enough wax
o Avoids chemical alteration
during
trimming
B. FREEZE-SUBSTITUTION Block after Insufficient impregnation Repeat
 Similar to freeze-drying in prep and cooling is impregnation
preservation moist and Re embed
crumbles
o Difference: Tissue fixed in Rossman’s
formula or 1% Acetone and dehydrated
in absolute alcohol