The document provides information about embedding and section cutting techniques in histopathology. It discusses the aims of embedding tissue, such as providing support and preventing distortion. Common embedding mediums include paraffin wax, resin, agar, and gelatin. Paraffin wax is the most popular due to being inexpensive, non-toxic, and allowing long term tissue storage. The document outlines the tissue embedding process and describes equipment used, including embedding molds and the Tissue-Tek system. Tissue orientation in the block is important for visualizing the desired morphology.

1. EMBEDDING AND SECTION
CUTTING
Presented by Dr.Sindhu
1st year postgraduate
Guided by Dr.krishna kumari
professor

2. HISTOPATHOLOGY TECHNIQUES
• Fixation.
• Tissue processing.
• Embedding.
• Microtomy or section cutting.
• Staining.

3. Introduction
• Embedding is the process of creating tissue blocks by using an external
support medium to enable microtomy.
• The embedding medium should be completely compatible with the infiltrating
medium in order to prevent tissue separation and to facilitate sectioning.

4. CONTENTS
• Aims
• Embedding medium.
• Types of moulds.
• Tissue embedding method.
• Tissue orientation.
• Tissue marking.

5. Aims of embedding
• To give support of the tissue.
• To prevent distortion of the tissue during cutting.
• To preserve the tissue for archival use.

6. Embedding medium
The choice of embedding medium depends upon
• Type of tissue.
• Type of microtome .
• Type of microscope.

7. Ideal impregnating medium
• Miscible with clearing agent.
• Liquid in higher temperature (30 to60*c) and solid in room temperature.
• Homogenous and stable.
• Non toxic and cheap.
• Transparent.
• Fit for sectioning the tissue.

8. The time duration and number of changes required for the impregnation in tissue
depends on;
• Type of tissue : The density of the tissue and the embedding medium should be
close otherwise tissue may not be sectioned properly and tissue will be
deformed.Hard tissue such as bone and cartilage takes more time for embedding
than soft tissue.
• Size of tissue : Thicker large tissue takes more time to impregnate with the
embedding medium. It also contains more clearing agent to remove.
• The type of clearing agent : Xylene and toluene are easy to remove than
cedarwood oil.
• Type of processing : Vaccum embedding enhances impregnation.

9. Various embedding mediums:
• Paraffin wax
• Ester wax
• Resin
• Agar
• Gelatin
• Celloidin

10. Paraffin wax
• Paraffin wax is a solid polycrystalline hydrocarbon and is produced as by-
product during refining of crude petroleum.
• This is the most popular universally accepted embedding medium for tissue
processing.
• This is non toxic and inexpensive medium.
• Melting points ranging from 52 to 62*c is used in our laboratory.
• Total 3 to 4 hour time in paraffin wax is sufficient for impregnation of tissue by
wax.

11. • Advantages of paraffin wax :
1. Tissue block can be stored for longer duration.
2. Non toxic .
3. Safe.
4. Cheap.
• Disadvantages of paraffin wax :
5. It may cause shrinking and hardening in case of prolonged impregnation.
6. Takes long duration for bone and eye impregnation.

12. Additives and modification of paraffin wax
To alter the physical characteristics of paraffin wax, the following modifications may be done :
• To increase hardness : addition of steric acid.
• Reduction of melting point : addition of phenanthrene.
• Improving adhesiveness with tissues and wax : addition of 0.5% of Ceresin.
• Dimethyl sulphoxide(DMSO) : The addition of small amount of DMSO in
paraffin wax reduces the infiltration time and removes the residual clearing
agent.It produces a homogenous matrix and better support.

14. Modified paraffin waxes
• PARAPLAST :
• Combination of highly purified paraffin wax and plastic polymers.
• Has greater degree of elasticity.
• Minimises tissue compression due to sectioning and gives wrinkle free serial
sections to be cut with 4mm ease.
• PARAPLAST PLUS :
• Wax of highest purity containing DMSO.

15. Paraffin wax is unsuitable medium when :
• Processing agents remove or destroy tissue components which are object of
investigation like lipids.
• Use of heat may have adverse effect on the tissue component.Eg..enzymes.
• Require thinner sections.Eg.electron microscopy.
• Infiltrating medium is not hard enough to support the tissue.Eg..undecalcified
bone.

16. Ester wax
• Harder than paraffin wax although it has low melting point.
• Can be compressed , so less likely to crumble while cutting.
• Thin sections are easily cut.
• Because of hardness of wax , sections from blocks need to be cut slowly with
a very sharp knife.
• POLYESTER WAX :
• Low melting point.
• Reduces heat induced artefacts - recommended for heat labile tissues.

17. Resin embedding
• Resin is much harder material than paraffin wax and these properties are
exploited either to cut hard materials such as undecalcified bone ,or to cut
thin that is 0.5 to 1.5 mm sections.
• For practical purpose two groups of resins may be employed : Epoxy resin
and Acrylic resin.
• To be a effective resin must have two phases :
1. Liquid phase with which the tissue can be infiltrated.
2. Solid phase which will support the tissue during microtomy.

18. Epoxy resin
• Epoxy resin is mainly used in election microscopy as it provides better
resolution and greater details of tissue.
• The high mechanical strength and particularly low shrinkage together with the
ease and consistency of polymerisation led to applications in light microscopy.
• The health hazards associated with this resin is slightly greater than with
acrylic resins, contact dermatitis is being a serious problem.
• Another limiting factor in the use of epoxy resin is having high viscosity and
consequent difficulties in impregnation, however low viscosity epoxy resins are
also available now.

19. Acrylic resins
• These are monomers of acrylic and methacrylic acids.
• Methacrylate monomer is available in the market along with hydroquinone
which should be removed by treating with weak alkali solution followed by
thoroughly washing with water.
• The presence of water may lead to small bubbles within the block.

20. Agar gel
• Agar gel helps in cohesion of friable and fragmented tissue particularly in
cytology sample ,endometrial curetting and small endoscopic biopsies.
• It does not provide good support of the tissue for section cutting.
• Agar-paraffin wax double embedding is more suitable technique than agar
alone.

21. Agar paraffin wax double
embedded block

22. Gelatin
• It is also used in small friable tissues and frozen section containing friable and
necrotic tissue.
• The melting point of gelatin is 35 to 40*c, and this low melting point makes it
unsuitable for embedding.
• The disadvantage of this method is that when staining techniques are applied
the gelatin tends to hold the stain, giving an indifferent background to the
section.

23. Celloidin medium
• Celloidin is Low Viscosity Nitrocellulose was mainly used for embedding
hard tissues.
• Disadvantages:
1. It is very difficult to cut sections thinner than 10 mm.
2. Processing is slow, impregnation and subsequent hardening of the block
taking weeks to complete.
3. Blocks and sections must be stored in 70% alcohol and preferably in the
dark, this is both hazardous and space consuming.
4. The reagents are highly inflammable.
5. As it is impossible to prepare ribbons of sections, it is a tedious process
to prepare serial sections.

24. Double embedding
• The preparation of serial sections of Celloidin impregnated tissue is normally a
tedious procedure since the sections will not adhere to one another in the
same manner as paraffin embedded material.
• Double embedding is the name given to a technique whereby the tissue is
first impregnated with Celloidin and subsequently blocked in paraffin wax .
• In this manner the advantages of both materials are to some degree are
combined.

25. L-Shaped Leuckhard embedding moulds
• It has 2 arms .
• The two L shaped are adjusted to make a
convenient size for block.
• Adequate lubricant such as glycerine is applied to
the L arms and metal plate for easy removal.
• The molten wax is poured in the space between
two arms and the tissue is placed within the
bottom of the liquid wax.
• The wax is subsequently cooled,and the block with
tissue in one surface is removed for microtomy.
Types of moulds

27. Stainless steel mould
• The mould is made up of stainless steel.
• The base of the mould is flat and well polished
that helps to remove liquid paraffin.
• The mould can be covered by a plastic ring.

28. Plastic mould
• Made up of chemical resistant
plastic.
• Most of the laboratories use
plastic embedding rings now.
• These are relatively inexpensive,
convenient & support the block
during sectioning.
• Cassettes are thin so less wax is
required.
• The space required for filling the
block is less.

29. • ICE TRAYS :
• Made of plastic .
• Each tissues blocked into individual compartment.
• PAPER BOATS :
• Cheap and convenient alternative.
• Best prepared from glossy paper.
• Tissue stored with identifying number written on projecting tag.

30. Tissue embedding method
• The following things are needed for tissue embedding:
1. Molten paraffin wax
2. Mould with cover
3. Metal plate to work

31. Method of embedding
• Open the tissue cassette , check requisition form to ensure the correct
number of tissue pieces is present.
• Select the mould, there should be sufficient room for the tissue with
allowance for at least a 2mm surrounding margin of wax.
• Fill the mould with paraffin wax.
• The tissue is transferred rapidly to the wax containing based mould with a pair
of warm forceps.
• Care should be taken that the forceps are not too hot and that excessive
pressure is not used.
• Ensure that no air bubbles are trapped and place the tissue with the surface
to be sectioned down against the base of the mould.

32. • Allow the wax in the bottom of the mould to solidify to hold the tissue in
place.
• The block should be immediately transferred to a refrigerated surface, a tray
of ice or once a skin has formed on the surface immersed in a trough of cold
water.
• Once solidification is complete the block may be removed from the mould.

33. Tissue - Tek system
• It is a combination of :
1. Dispenser of liquid paraffin in a constant temperature.
2. A metal plate to make the tissue block.
3. Cold plate.

36. Tissue orientation
• Ability to see desired tissue morphology is dependent on the correct placement or
orientation of the sample in the block.
• Products which assist in correct orientation are :
1. Marking systems.
2. Tattoo dyes.
3. Biopsy bags.
4. Sponges & papers.
• Orientation of tissue - offer least resistance of the tissue against knife during
sectioning.

37. • The correct orientation of the tissue is very important for proper cutting and
microscopic examination.
• Some of the tissue needs special care as described below :
1. Tubular tissue like fallopian tube , vas deferens ,artery etc.. should be sectioned
at right angles so that the lumen is clearly visible.
2. Tissue with epithelial surface should be placed vertically and right angle to the
surface so that we can get all the layers.
3. Multiple section of tissue such as endometrial curetting should be placed in
central position.
4. Linear long tissue should be placed diagonally.
5. Muscle biopsies should be embedded so that both longitudinal and transverse
planes are obtained.
6. Long membranous tissue such as amniotic membrane should make as Swiss roll.

39. 40
• The tissue marking by ink is needed for the following purposes :
1. To identify the resection plane or outer margin of the tissue.
2. To help in embedding the tissue.
3. Any area of interest to identify such as the area of transitional zone in cone
biopsy of cervix etc..
Tissue marking

40. Characteristic features of tissue markers :
• Should not be dissolved in fixative and tissue processing agent.
• Should not penetrate deeper tissue.
• Should be recognisable in stained section both microscopically and
macroscopically.

41. Common tissue markers :
• India ink .
• Silver nitrate.
• Rose Bengal.

42. Process of application :
• Clean and dry the tissue with blotting paper
• Apply dye with cotton swab
• Dry it
• Put fixer over dye (3% acetic acid or 50% white vinegar).
• Dry the specimen with sponge.
• Process.

44. MICROTOMY

45. MICROTOMY
• The means by which tissue can be
sectioned and attached to a surface for
further microscopic examination.
• Microtome is a mechanical device for cutting
thin uniform slices of tissue sections.

46. Types of microtomes
Based on the mechanism
• Rotary Rocking
• Rotary
• Base-sledge
• Sliding
• Cryostat
• Ultra

47. Rotary microtome

48. Types of rotary microtome
• Manual: Completely manipulated by the operator.
• Semi-Automated: One motor to advance either the fine or
coarse hand-wheel.
• Fully Automated: Two motors that drive both the fine and the
coarse advance hand-wheel

49. Advantages of Rotary microtome
• Ability to cut thin 2-3mm sections.
• Large and heavier knife used-less vibration when cutting hard tissue.
• Cutting angle of knife is adjustable.
• Easy adaptation to all types of tissues (hard, fragile or fatty)sectioning.
• Can cut celloidin-embedded sections by using a special holder to set
the knife obliquely.
• Ideal for cutting serial sections, large number of sections from each
block.

50. Parts of a Rotary microtome

51. • Knife holder base: A part that anchors the knife holder to the
microtome stage. The knife holder base can be moved
toward or away from the block, but must be stationary and
locked during microtomy.
• Knife holder: this part is comprised of several components
including the blade clamp that holds the blade, the knife tilt
for adjusting the knife angle, and the face plate that guides
that ribbons away from the blade and towards the operator.
• Coarse handwheel: Moves the block holder either toward
the knife or away from the knife.
• Micron adjustment: Micron settings for section thickness can
range from 1 to 60 microns on most microtomes.

52. • Advancement hand wheel: Turns in one direction and advances the
block toward the knife at the specified microns.
• Safety Lock: Most handwheels are equipped with a safety lock to
prevent the wheel from releasing and having the block holder come
down towards the blade while a block is inserted or removed. It should
be used anytime the microtomist is not actively sectioning paraffin
blocks.

53. Base sledge microtome:
• Originally designated for cutting sections of
very large blocks(whole brain)
• In laboratories where very hard tissue or
large blocks are usual, this type of
microtome is favored
• It is most commonly used in neuropathology
and ophthalmic pathology
• Here the specimen is held stationary and the
knife slides across the top of it during
sectioning

54. Rotary rocking microtome:
• It has the advantage of producing a
flat face to the tissue block.
• Most of them have a retracting
mechanism which takes the tissue
block away from the knife on the
upward stroke.
• Although they can be used for
paraffin wax work they are used
more commonly in cryostats

55. Sliding microtome:
• The knife or the blade is stationary, the
specimen slides under it during
sectioning.
• It was designed mainly for cutting
cellodin-embedded blocks of tissue.
• It can also be used for paraffin wax-
embedded sections

56. Ultra microtome:
• These are used exclusively for electron
microscopy
• Prepare ultra thin sections.
• It has been reported that sections can be
cut as thin as 10 nm.
• Knives are usually made from glass,
diamond or sapphire.
• The block is brought to the knife edge
under microscopical control and as
each section is cut it is floated on to a
water bath adjacent to the knife edge

57. Paraffin section cutting
Equipment required
• Water bath
• Slide drying oven or hot plate
• Fine pointed or curved forceps
• Sable or camel haired brush
• Scalpel
• slide rack
• Clean slides
• Teasing needle
• Ice tray or cooling platform
• Chemical-resistant pencil or pen

58. Floatation(water) bath
• A thermostatically controlled water bath
is used for floating out tissue ribbons
after sectioning.
• The temperature of the water should be
about 10 degree Celsius below the
melting point of the paraffin wax.
• Care should be taken to prevent water
bubbles from being trapped under the
section and this can be accomplished by
using distilled water in the bath.
• Alcohol or small drop of detergent may
be added to the water to reduce the
surface tension allowing the section to
flatten out with ease

59. Drying oven or hot plate
• With a temperature setting at the melting
point of the wax no obvious damage is done
to the sections and drying is complete in 30
minutes.
• Too hot temperature may cause dark
pyknotic nuclei or nuclear bubbling and loss
of nuclear detail.
• For delicate tissues a lower temperature is
desired for drying so as to avoid splitting and
cracking of the section due to excess heat 37
degrees Celsius for 24 hours is
recommended.
• Time for drying slides should be monitored
as many immunohistochemical or special
stains are heat sensitive.

60. Brush and forceps
These instruments are necessary for the handling of sections during cutting
and for the removal of folds and creases formed in the sections during
floating out

61. slides
• For normal routine work 76x25mm slides are
universally used.
• 1.0-1.2mm thick slides are preferred because
they do not break easily.
• Large size of slides are used for sections of eyes
or CNS tissues.
• Chemical-resistant pens and pencils are routinely
used to label the slide.
• Automatic slide labelling machines are now
available.
• Slides which are positively charged or pre-treated
with an adhesive resist detachment of the tissue
from the slide during staining

62. Section adhesives
• Most commonly used adhesive is albumin.
• Others are starch paste and chrome gelatin.
• Albumin solution is prepared by mixing equal parts of glycerine, distilled
water and white of eggs, then filtered through coarse filter paper and a
crystal of thymol is added.
Disadvantages
Albumin retains some of the stain and gives a dirty background.
Thymol resistant organisms growing in the adhesive have been known to cause
confusion in a gram stained section.

63. There are occasions when sections may tend to float from the slide
and these are:
1. When sections are submitted to strong alkali solutions during
staining.
2. Cryostat sections for immunofluorescence,
immunocytochemistry and urgent diagnosis.
3. Tissues from the CNS
4. When sections are submitted to high temperatures.
5. Tissues containing blood clot.
6. Tissues which have been decalcified.

64. Poly-L-lysine
• This is brought as a 0.1% solution which is further diluted for
use(1 in 10 with distilled water). Sections are coated with
this dilute poly-L-lysine and allowed to dry.
• The ability of this substance to stick the section to the slide
slowly loses its effectiveness. Therefore the coated slides
should be used within a few days.

65. 3-aminopropyltriethoxysilane(APES)
• This is by far the best section adhesive available and coated slides can
be stored for a along time. Slides are dipped in 2% APES in acetone
drained then dipped in acetone, drained again and finally dipped in
distilled water.
• Invaluable in cytology particularly for cytosine preparation of
proteinaceous or bloody material.

66. Charged or plus slides
• Permanent positive charge by coating the slide with a basic
polymer in which a chemical reaction occurs, leaving the amino
groups linked by coavalent bonds to the silicone atoms of the
glass.
Advantages
• Superior resistance to cell and tissue loss during staining or
pre-treatments such as enzyme and antigen retrieval

67. Practical section cutting
BLOCK TRIMMING
• Wax is removed with a sharp knife until 1/8th inch remains on all sides of the
tissue.
• Only small flakes of wax should be trimmed at a time
• Attempts to trim large pieces can lead to splitting and exposure of tissue.

68. TECHNIQUE OF CUTTING
• Insert the knife in the knife-holder &screw it tightly
• Fix the block in the block holder and ensure it is
secure
• Feed mechanism is adjusted until the wax block is
almost touching the knife. Ensure that the whole
surface of the block will move parallel to the knife
so that straight ribbon of sections is obtained.
• All screws should be tight to avoid faulty
sectioning.
• For block trimming section thickness of 15microns
with a rough knife is taken
• Sharp knife is used for sectioning.
• Reset the thickness gauge to required
thickness.4-5 microns recommended for routine
work.

69. • Apply ice to the block surface to make the wax hard
which would have become soft by frictional heat.
• There should be a smooth continuous plastic flow of
sections in the form of a ribbon
• When the ribbon comes off it is held gently with a
fined moistened brush or forceps and then
transferred to water bath.
• Section is then floated on the water bath(temp 5-6*
below melting point of wax )to remove creases.
• Clean or albuminized slide is half submerged in
water and the section is picked up using a
dissecting needle.
• The slide is then set in an upright position to drain.
• Slides are kept in incubation(37degrees overnight
for plain slides and 60degrees for 2 hours for
albuminised ones)

70. Cutting hard tissues
• Since the introduction of disposable blades cutting hard
tissues is now less difficult and the main reason for cutting
difficulties is more likely to be poor fixation or over
processing
• Prolonged melting ice treatment of the block or exposing the
block surface to running tap water for 30 minutes, will often
overcome almost every hard tissue
• A slight reduction in the knife slant may also yield results.
• If these remedies fail, softening fluids such as
mollifex(saturated into cotton,wool)can be used on the block
surface. this will penetrate the block by some 15-
20micrometer and therefore it is essential to retrieve the
immediate section.

71. Surface decalcification
• When a block has been trimmed to reveal the tissue surface,
small foci of calcium may occasionally be removed.
• The block can be removed from the chuck and placed face
down on a pad of cotton wool saturated with 10%HCL.
• After treatment for approximately 1 hour, the block is
relocated in the microtome and the first few sections can be
cut before calcified tissue is reencountered within the tissue

72. REFERENCES
1.Culling.C.F.A,allison.R.T,Barr.W.T,cellular pathology
techniques.4thedition;Butterworth & Co.Ltd,1985;58-71.
2. Kim.S, Layton.C, Bancroft.J.D, Bancfrot’s theory and practice of histological
techniques.8th edition; Elsevier: 90-91.

73. Thank you