1. Decalcify in the electrolytic apparatus in formic acidhydrochloric acid for 1 to 4 hours.
Electrolytic decalcifying solution
Distilled water to make 1000 cc
2. Wash in running water for 24 hours.
3. Dehydrate, clear, and embed.
Embedding in paraffin is accomplished most rapidly and
gives the best results when thin sections of soft tissues
are wanted. Since paraffin is not miscible with water, the
tissue must be dehydrated and then cleared in a solution
that is miscible with paraffin.
Automatic tissue processing units are usually employed
these days. Several manufacturers provide such units (Fig.
25.1). This instrument has several beakers, each filled
with a specific fluid in it. The basket containing the tissue
sections with identification tags is automatically dipped
and revolved in the beakers at preset timings. Usually,
paraffin-embedding process is started in the afternoon
and is complete by mornings on the following day. Various
schedules for paraffin processing are given below.
2. Alcohol 95%—2 changes 1–2 hours each
3. Alcohol absolute—3 changes 1–2 hours each
4. Xylene—2 changes 1–2 hours each
5. Melted paraffin—3 changes 1–2 hours each
6. Embed in paraffin and cool quickly.
1. Alcohol 80%—2 changes 1–2 hours each
2. Alcohol 95%—2 changes 1–2 hours each
3. Alcohol, absolute—3 changes 1–3 hours each
4. Chloroform—2 changes 1–2 hours each
5. Paraffin, melted—3 changes 1 hour each
1. Alcohol 80%—2 changes 1–2 hours each
2. Alcohol 95%—2 changes 1–2 hours each
3. Alcohol, absolute—3 changes 1–2 hours each
4. Benzene—1 change 1–2 hours each
5. Paraffin bath—3 changes 1 hour each
(In place of alcohol, one may use acetone. Xylene and
benzene can be used in place of one another. Benzene,
however, is carcinogenic and should be avoided).
FIG. 25.1: Automatic tissue processing unit
(Courtesy: Yorco Sales Pvt. Ltd)
It is important that the knife used for cutting sections
be very sharp and without nicks. A perfect edge for a
microtome knife may be defined in simple terms as the
junction of two smooth plane surfaces at an angle of
about 14°. Knife sharpening may be done by mechanical
means on commercial automatic knife sharpeners or
done manually called honing and stropping. Nowadays
disposable knife blades with appropriate blade holders
After the paraffin block has been secured at the appropriate
place in the microtome, adjustment of the block and the
knife is now required. Keep a piece of cotton in a dish of tap
water and an ice cube in a petridish beside the microtome
To facilitate sectioning, apply the wet cotton to the
surface of the block after rough cutting. Then place the ice
cube directly on the knife to flatten the side of the cube
that is to be applied to the surface of the block. Be sure to
crank the block back a fraction of a millimeter from the
knife edge, so that the first section cut after the block has
been soaked and chilled will not be too thick.
Collect the section ribbons in a bowl containing hot
water and unfurl or straighten the sections gently with a
The glass slides on which tissue sections are to be mounted
must be marked beforehand with the identifying case
number. A glass marking pencil is used for this purpose.
Paraffin sections may be attached to slides in several
ways. A small drop of Mayer’s egg albumin is smeared
over the surface of the slide with the finger and the excess
rubbed off with the heel of the hand, or it can be applied
with a clean foam rubber sponge. A sponge is usually
preferred so that the epithelial cells from the fingers will
not adhere to the slide and produce artefacts when stained.
Mix well and filter through coarse filter paper, or through
several thicknesses of gauze. Add a crystal of thymol to
Egg Adhesive from Dried Albumin
Filter on Buchner’s funnel with vacuum. To 50 cc of filtrate,
and 50 cc glycerin, add a crystal of thymol to preserve.
Slides smeared by one of the above-mentioned egg
adhesives are taken below the floating section and the
section is placed in the center of the slide; leave it to dry to
1. Fix small blocks of tissue in 10% formalin.
2. Wash blocks in water before freezing.
3. Put a drop of water on the holder and place the block
in position parallel to the knife edge.
4. Holding the block with the index finger or a glass slide,
turn on the coolant gas/fluid slightly. When block is
firmly fixed to holder, release more coolant gas/fluid
5. Start sectioning and continue until a complete section
is obtained. Usually, the block will have thawed to
about right consistency by this time. If it is frozen
too hard, the sections may shatter. Allow the block to
thaw slightly and try again. If it has become too soft,
the sections will also shatter or fracture. The correct
temperature can only be judged by experience.
Sometimes rubbing the finger across the block will
give it the right consistency for good sections to cut. It
6. Lift the sections off the knife edge with the tip of the
little or ring finger which has been dipped in distilled
water. Place in a Petri dish of distilled water. Dry
the knife between sections as water will cause the
following section to be uneven or perforated.
7. Frozen sections may be stained with polychrome
methylene blue, hematoxylin and eosin, or fat stains.
(Courtesy: Yorco Sales Pvt. Ltd)
794 Concise Book of Medical Laboratory Technology: Methods and Interpretations Staining
Staining may be done by the free flotation method or the
sections may be picked up on the slide first and stained as
Fat stains must be mounted in glycerin jelly. Sections
stained by other techniques may also be mounted in
glycerin jelly or they may be dehydrated, cleared in xylene
Removal of Pigments and Precipitates
If Zenker-fixed material stored for a long-time is to be
stained with alum hematoxylin, it will be found that areas
where the mercuric chloride is deposited stain deep
blue and distort the microscopic picture. Therefore, it is
1. Deparaffinize the sections by putting them on a hot
plate and then take through two changes of xylene,
absolute alcohol and 95% alcohol.
2. Place in alcoholic iodine (1 g iodine in 100 cc of 80%
alcohol) for 10 to 15 minutes.
4. Place in 5% aqueous sodium thiosulfate solution
5. Wash in running tap water for 10 to 20 minutes and
rinse well in distilled water before staining.
1. Deparaffinize the sections through two changes each
of xylene, absolute alcohol, and 95% alcohol.
2. Rinse well in distilled water.
3. Let stand in saturated aqueous picric acid solution for
4. Wash well in running tap water.
This picric acid solution will not bleach malarial pigment.
1. Deparaffinize the sections through two changes each
of xylene, absolute alcohol and 95% alcohol.
2. Rinse well in distilled water.
3. Place in bleaching solution for 5–10 minutes.
4. Wash well in running tap water, and stain as desired.
1. Deparaffinize the sections through two changes each
of xylene, absolute alcohol and 95% alcohol.
2. Rinse well in distilled water.
3. Place in 0.25% aqueous potassium permanganate
5. Place in a 5% aqueous oxalic acid solution or a
hydrobromic acid solution (HBr 1 part, distilled water
3 parts) until sections are clear (2–5 minutes).
6. Wash in running tap water for 10 minutes, rinse in
distilled water, and stain as desired.
1. Deparaffinize the sections through two changes each
of xylene, absolute alcohol, and 95% alcohol.
2. Wash well in distilled water.
3. Place in a 5% aqueous solution of ammonium sulfide
4. Wash in running tap water for 15–20 minutes, rinse
well in distilled water and stain as desired.
1. Deparaffinize the sections through two changes each
of xylene, absolute alcohol, and 95% alcohol.
2. Place in a saturated alcoholic picric acid solution or 1
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