Decalcification - Slower action, greater tissue distortion

- Good nuclear staining

 Some tissues contain calcium deposits which are *rapid proprietary soln- w/ HCl
extremely firm and which will bot section *slow proprietary soln.- w/ buffered
properly with paraffin embedding owing to the formalin/formic acid
difference in densities between calcium and - Vin Eibner’s fluid- NaCl, HCl, H2O
paraffin  Good cytologic staining
 Bones, teeth, calcified tissues-tuberculous lungs, 3. Formic acid
arteriosclerotic vessels - Better nuclear staining with less tissue distortion $*
 Poor cutting hard tissues/knife damage safer to handle than nitric and HCl
- Know ptx case- if too large- use saw - 2-7 days slow
- Fixative and decal agent
To remember:
- Excellent nuclear and cytoplasmic staining
 Change decalcifying agent regularly  Formic acid- Na citrate soln
 “grating” sensation during cutting=place block in ( better nuclear staining than nitric acid)
10% HCl for 1 hr 4. TCA
 Rapid decalcification- produces effect on nuclear - 4-8 days very slow acting
staining- (failure of nuclear chromatin to take up - Good nuclear staining
hematoxylin) - Weak
Methods: 5. Sulfurous
 Acid decalcification - Weak
 Chelating agents - For minute pieces of bone
- EDTA-combine with Ca to form salts 6. Chromic acid (flemmings fluid)
 Ion exchange resins - Chromic acid, osmium tetroxide, glacial HAc
- Ammonium form of Polysterene resin - Fixative and decalcifier
 Electropheresis - Nuclear staining with hematoxylin is inhibited
- Forms precipitate at the bottom * carcinogenic,
I. Acid Decalcification corrosive to skin
7. Citric Acid – citrate buffer pH4.5
 Nitric acid-most common
- With zinc sulfate and chloroform
 HCL- Surface decalcification
- 6 days, slow
 Formic acid-mod. Decal for research
- Good nuclear and cytoplasmic staining
 Trichloroacetic avid- fix and decal
 Sulphurous acid- small pcs of bone Procedure:
 Chromic acid- fix and decal  Specimens should be decalcified in hydrochloric
 Citric acid- no cell distortion acid/ formic acid working soln 20 times their
 Von ebners’ fluid- NaCl+ HCl, for teeth and small volume
pcs of bone  Change to fresh soln. Each day until
decalcification is complete
1. Nitric acid o It may take 24 hrs up to days or mos
- Most common for routine tissue processing (Aq depending on size of the specimens
nitric acid 10% 12-24 hrs) o Once the decalcification is complete,
Example: times specimens in water briefly and
▪ Formol-nitirc acid transfer ammonia soln to neutralize acid
- Rapid acting left in specimens for 30 min
- Yellow nitrous acid formation- 5% Na sulfate and  Wash specimens in running water tap water
water or 0.1% urea thoroughly up to 24 hrs
▪
 Routine paraffin embedding
Pereyni’s fluid
- + chromic acid and abs ethyl
II. Chelating Agents
- Slow decal
EDTA (versene)
▪ Phloroglucin- Nitric Acid
- Excellent staining
- + Phlorglucin
- Very slow
- For urgent works
- Slight tissue hardening produced
- Complete decalcification cannot be determined by
III. Ion exchange resin
chemical
- *artifacts produced, usually caused by CO2 bubbles
2. HCl
- Slow
 IV. Electrophoresis ( Electrical ionization)  Heat hastens decal, but increases damaging
Electrophoresis effect of acid to tissues
- Formic acid, conc HCl, distilled water  Mechanical agitation, sonication
 Ideal time -24-48 hrs.
Measurement of Decalcification status
 Physical/mechanical
 X-ray/Radiological
 Chemical-litmus paper > red if due to acidity, add
NH3 drop by drop> blue litmus; if cloudy -still w/
calcium; if clear:+ ammonium oxalate, 30 mins>
cloudy if incomplete

Procedure:
 Insert a pipette into the decalcifying soln
containing the specimen
 Withdraw approx 5 Ml of the HCl/formic acid
decal soln. From under the specimen and place it
in a test tube
 Add approx 10 ml of the NHO3/ ammonium
oxalate working soln., mox well and let stand
overnight
 Decal is complete when no ppt is observed on 2
consecutive days of testing....

Physical test:
 The physical tests include bending the specimen
or inserting a pin, razor, or scalpel directly into
the tissue
 The disadvantage of inserting a pin, razor or
scalpel is the introduction of tears and pinhole
artifacts
 Slightly bending the specimen is safer and less
disruptive but will to conclusively determine of all
calcium salts have been removed
 After checking for rigidity, wash thoroughly prior
to processing

Tissue softeners
 Perenyi’s – 12- 24 hrs.
 4% Aq phenol-1-3 days
 Molliflex (swollen and soapy appearance)
 2% HCl
 1% HCl in 70% alcohol

Post decalcification
 Remove acid by sat lithium carbonate soln or 5-
10 % Aq NaHCO3 for several hrs
 Running tap water
 If EDTA is used-use 70% Alcohol

Rate of decalcification
 More con acid soln.-more rapid but more harmful
to tissue
 DEHYDRATION 2. Excellent for slow processing
 The removal of water 3. Miscible with paraffin
 In tissues,water is present in both free and bound
Disadvantages:
forms and needs to be removed before
processing can cont. 1. Odorous
 Dehydratiom is usually carried out using alcohols 2. Long periods of infiltration necessary
( such 3. Dehydrating power low
 Although dehydration can also cause tissue
shrinkage, the stage is necessary in all infiltration
methods, except where tissues are supported by Tertiary butanol (butyl alcohol)
an aqueous embedding medium (such as water Advantages:
soluble waxes)
 Boiling point:82.8 C
 In paraffin was processing, dehydration from
1. Universal.solvent-act as dehydrant and
aqueous fixatives such as formalin is usually
clearing agent
initiated in 70% alcohol before progressing
2. May be used in staining series as a
through 90%-95% to absolute alcohol before
dehydrant
proceeding to the clearing stage
3. Mixes with water, ethanol, xylene and
 Direct transfer to 95% alcohol is often performed paraffin in all proportions
of tissues are adequately fixed.
Disadvantages:

1. Odorous
Acetone 2. More expensive than butanol
Advantages: 3. Prima5y infiltration muse be done in half tertiary
butanol and half paraffin, prior to paraffin
 Boiling point:56 C impregnation
1. Rapid dehydration 4. Reagents tends to solidify at room temperature
2. Less expensive than ethanol or below 25C
3. Does not extract methylene blue and
other dyes from stained sections Ethanol (ethyl alcohol)
Advantages
Disadvantages:
 Boili g point:73.3C
1. Requires a clearing agent 1. Nontoxic
2. Volume must be 20 times that of tissue 2. Miscible in all proportions with water
3. Best processing requires a graded series of a 3. Little shrinkage if graded alcohols are
mixture of acetone and xylene before one can go used
into paraffin 4. Can be used.on eyes and embryos, if
4. Needs good ventilation: evaporates rapidly; graded alcohols are used
flammable 5. Fast acting
6. Still considered best dehydrant
Alcohols 7. Reliable

 These are clear, colorless, flammable and Disadvantages:

hydrophilic liquids that are miscible with water 1. Expensive
and most of the organic solvents. In addition to 2. Avoid long periods in absolute ethanol.to prevent
their role as dehydrants,alcohol also act as excessive shrinkage and hardening
secondary coagulant fixatives during tissues 3. May be difficult to obtain
processing. The most commonly used alcohol 4. Extracts methylwn blue and other thiazine.dyes
used in tissue processing is ethanol from secretions

Isopropanol
Butanol Advantage:
Advantages: Boiling point: 82.3 C
 Boiling point: 117.7C 1. Excellent substitute for ethanol
1. Less shrinkage and hardening than with 2. Less shrinkage and hardening than ethanol
ethyl
 3. Sufficiently water free to use in place of absolute high molecular weight polyethylene glycols are
ethanol solid and can be used for embedding tissues
4. Lilie consider it “the best all-around Substitute for
ethyl alcohol”
5. Less expensive
Cellosolve (Ethylene glycol monoethyl)
Advantages:
Disadvantage:
Boiling point: 156.4 C
1. Cannot be used in the celloidin technic since
1. Rapid dehydrant
nitrocellulose is insoluble in it
2. Tissue may remain in it for months without injury
2. Cannot be used for preparing staining solutions,
3. Avoids distortion and does not requires graded
since dyes are not soluble in it
dilutions

Disadvantages:
Methanol
1. Expensive
 The reagent is a good ethanol substitute but
2. Rapidly absorbs water from the air
rarely used because it is volatile, flammable and
3. Requires clearing agent
costly. Methanol tends to harden tissues more
than ethanol and is a poor lipid solvent

Phenol Tetrahydrofuran-THF
 This consists of clear hygroscopic crystals and is Advantages:
also available in a liquefied form. Phenol is
soluble in water, alcohol and most organic
solvents. However, phenol develops a pink
discolour on exposure to air and light so
containers must be protected from light and
tightly sealed.

Pentanol (amyl alcohol)

Advantage:
Boiling point: 128 C

1. Miscible with 90% alcohol, toluene and xylene

2. Dissolves paraffin wax Dimethoxypropane (DMP) and diethoxypropane (DEP)

Disadvantages:  These are flammable, miscible with wax and are

used for the chemical dehydration of tissues.
1. Toxic They are suitable for rapid manual processing or
2. Cannot be used in poor ventilated rooms machine processing and are comparable to
3. Not miscible in water conventional dehydration for tissue morphology
and staining reactions

Dioxane (diethylene dioxide)

Polyethylene glycols Advantages:
 These are used to dehydrate and embed tissues Boiling pint: 101.5
that are affected by the solvents and heat of the
paraffin wax method. They are clear, viscous Refractive index: 1.42
liquids or solids of low toxicity. At low molecular
1. Universal solvent-dehydrates and clears
weight the polyethylene glycols are liquid and can
2. Miscible with water alcohol, xylene and paraffin
be used to dehydrate the tissues as they pass
3. Does not harm tissue over long time periods
thro9ugh glycols of increasing molecular weight
4. Produces less shrinkage than ethanol
and viscosity. However, at room temperature
5. Faster dehydrant than ethanol
Disadvantages:

1. Needs large volume for dehydration

2. Costs about for times more than does absolute
alcohol
3. Must be used in well-ventilated room.
4. Cumulatively toxic
5. Odorous
6. Distort tissue-containing cavities

Triethyl Phosphate

Advantages:

Boiling point: 215 C

1. May be used in routine technic

2. Displaces water readily with slight distortion
3. Does not harden tissue excessively
4. May be used as a dehydrant in the staining
sequence
5. Soluble in alcohols, benzene, toluene, xylene,
ether, chloroform

Additives to dehydrating agents

 4 % phenol added to 95 % ethanol- softens hard

tissues
 Hard tissues- immerse in glycerol/alcohol mixture
or in Molliflex