Tissue processing involves removing water from tissue and replacing it with paraffin wax to provide rigidity for microscopic examination. The main steps are fixation, dehydration using increasing concentrations of alcohol, clearing with xylene to remove alcohol, and impregnation with molten paraffin wax. Automated tissue processors complete this process overnight using different stations for each step. Factors like tissue size, agitation, heat, and vacuum pressure influence effective processing. Ethyl alcohol is most commonly used for dehydration, while xylene is used for clearing prior to paraffin wax impregnation and embedding.

1. Tissue Processing, embedding
and embedding medias

2. Tissue Processing
• Aims of tissue processing:
– To provide sufficient rigidity to the tissue so that
it can be cut into thin section for microscopic
examination.
• Principle of processing:
– In tissue processing the water within the tissue is
removed, and another medium (usually paraffin
wax) is impregnated in the tissue that provides the
adequate support to the tissue.

4. Tissue processors
• Processes of dehydration, clearing and impregnation
are carried out in a composite equipment which is
known as automated tissue processor.
• Automated tissue processor can be:
• Open (hydraulic) system
• Closed (vacuum) type

5. Open (hydraulic) tissue processor
• It has 12 stations—10 stations are glass/steel jars and
2 stations have thermostatically controlled wax bath.
• These jars are used as follow:
– For fixation in formalin: 1 jar.
– For dehydration in ascending grades of alcohol: 6
jars, one each of 70%, 80%, 90% and 3 for 100%.
– For clearing in xylene: 3 jars.
– For impregnation in molten paraffin wax: 2 wax
baths.

8. • Tissue moves automatically by hydraulic mechanism
from one jar to the next after fixed time schedule as
set in the program.
• Generally, 1.5 hours duration is given at each station
and whole process takes about 18 hours (overnight).
• For rapid processing (biopsy), modern systems have
programmes for short run in which entire tissue
processing is completed in maximum of 2.5 hours;
tissue stays at each station for 10-20 minutes.

9. Closed (vacuum) tissue processor
• Tissue cassettes are placed in a single container
while different processing fluids are moved in
and out sequentially according to electronically
programmed cycle.
• It has the advantage that there is no hazard of
contamination of the laboratory by toxic fumes
unlike in open system.
• In addition, heat and vacuum shorten the
processing time. Thus, it can also be applied for
short schedules or rapid processing of small

11. Factors that Influence Tissue
Processing
• Size of the tissue:
– The smaller the size, the better the processing.
– Optimum thickness of the tissue - 3–4 mm only.
• Agitation:
– The tissue gets better contact with the
surrounding medium if it is completely immersed
and gently agitated.
– Too rapid agitation may damage the soft and
delicate tissue

12. Factors that Influence Tissue
Processing
• Heat:
– Increases the better penetration of fluid.
• Viscosity:
– The higher the viscosity of the medium, lower the
penetration.
– Heat reduces the viscosity of the medium and
helps in better penetration.
• Negative pressure (Vaccume):
– Negative pressure removes trapped air in the
tissue.

14. Dehydration –Removal of water
• The process of removing intercellular and
extracellular water from the tissue following
fixation and prior to wax impregnation is known
as "dehydration”, .
• Solutions utilized for this process are called
"Dehydrating Agents“.
• Sharp difference of concentration gradient of the
dehydrating fluid may damage the delicate tissue.
• Gradual dehydration is necessary from low to
high concentration of dehydration fluid. Routine
laboratory: 70, 90 and 100% alcohol for 1.5 to 2 h

15. • Common dehydrating agents:
– Ethyl alcohol
– Methylated spirit
– Methanol
– Butyl alcohol
– Isopropyl alcohol
– Dehydrating agents other than alcohol: dioxane,
ethylene glycol and acetone

16. Ethyl alcohol
• Most popular and most commonly used
dehydrating agent.
• This is a clear , colourless and flammable fluid.
• It is considered to be the best dehydrating agent
because :
– fast-acting,
– mixes with water and many organic solvents
– penetrates tissues easily.
– Little shrinkage if graded alcohols are used
– Non- poisonous
– Not very expensive .

17. • As a dehydrating agent ethyl alcohol is used in
50, 70, 90 and 100% concentration.
• For delicate tissue, the dehydration may be
started from 30% concentration of ethyl
alcohol.
• If tissue is immersed in the ethyl alcohol for
long time, then the removal of attached water
from the carbohydrate and protein molecules
causes hard and brittle tissue

18. Anhydrous Cupric Sulphate in Final
Container
• Anhydrous cupric sulphate (CuSO4) is a white
powder that draws water from the alcohol and
thereby helps in dehydration.
• About 1 cm layer of this powder is kept in the
bottom of the container.
• The cupric sulphate powder should be
covered with two to three layers of filter
paper to prevent any colouring of the tissue.
• When the CuSO4 becomes hydrated, the
colour of the powder changes to blue. This
gives warning signal to change the alcohol and

19. • Advantage:
– Increases the life span of alcohol
– Better dehydration
– Good indicator to change alcohol

20. Methanol
• Clear, colourless, volatile and inflammable
liquid.
• It can be used as a substitute of ethanol, but
it is rarely used in laboratory because of its
volatility and high cost.

21. Butanol (butyl alcohol)
• Boiling point 117.7° C
• Advantages:
– Less shrinkage and hardening than with ethyl
alcohol
– Excellent for slow processing
– Miscible with paraffin
• Disadvantages:
– Odorous
– Slow-acting
– Long periods of infiltration necessary
– Dehydrating power low

22. Isopropyl Alcohol
• Available as isopropanol (99.8%).
• This is miscible with water and liquid paraffin.
• It is a relatively rapid acting, non-toxic .
• causing minimal tissue shrinkage and
hardening.
• No government restrictions on its use.
• It is a good lipid dissolving solvent
• Less expensive than alcohol

24. Clearing
• Next step of processing after dehydration.
• Removal of dehydrating agent from the
tissue because many dehydrating agents are
not miscible with the impregnating material
(paraffin wax).
• The clearing agent should be miscible with
both the dehydrating agent and the
embedding medium.
• The refractive index of the clearing agent is
similar to the tissue, and therefore it gives

25. Aims of clearing
• Removal of dehydrating agent (e.g. alcohol)
to facilitate impregnation of paraffin wax .
• To make the tissue clear and improve the
microscopic examination

26. Ideal clearing agent:
• It should be miscible with alcohol to promote rapid removal of the
dehydrating agent from the tissue.
• It should be miscible with, and easily removed by melted paraffin wax
and/or by mounting medium to facilitate impregnation and mounting of
sections.
• It should not produce excessive shrinkage, hardening or damage of tissue.
• Low viscosity and high penetration rate
• Low melting point : easily and quickly removed by the molten wax
• It should not evaporate quickly in a water bath.
• It make tissues transparent.
• Less toxic and Less inflammable
• Cheap

27. • Volume of clearing agent: 40 times the volume
of the specimen
• Total duration:
– Smaller biopsy: 1 h
– Larger tissue: Three changes in xylene or toluene
60 min each
• End point detection: Tissue becomes
transparent
• Prolonged exposure to clearing agent: The
brittle and more friable tissue
• Different clearing agents: Xylene, toluene,
chloroform, amyl nitrate, cedarwood oil and

28. Xylene
• Most commonly used clearing agent in the
laboratory.
• This is a clear and inflammable liquid.
• The small pieces of tissue are cleared rapidly
by xylene within 30–60 min.
• Prolonged exposure to xylene may make the
tissue hard and brittle

29. Advantages:
• Rapid clearing agent, suitable for urgent biopsies
which it clears within 15-30 minutes.
• It makes tissues transparent.
• miscible with absolute alcohol and paraffin.
• For mounting procedures, it does not dissolve
celloidin and can, therefore, be used for celloidin
sections.
• It evaporates quickly in paraffin oven and can,
therefore, be readily replaced by wax during
impregnation and embedding.

30. Disadvantages
• It is highly inflammable and should be
appropriately stored.
• If used longer than 3 hours, it makes tissues
excessively hard and brittle.
• Xylene becomes milky when an incompletely
dehydrated tissue is immersed in it.
• Xylene may irritate eyes, nose and respiratory
tract.
• It can be absorbed through the skin and cause
dermatitis.
• At high concentrations, it is toxic and narcotic.

32. Impregnation
• Process whereby the clearing agent is
completely removed from the tissue and
replaced by a medium that will completely fill
all the tissue cavities and give a firm
consistency to the specimen.
• Allows easier handling and cutting of suitably
thin sections without any damage or
distortion to the tissue.

33. • Ideal impregnating medium:
– Miscible with clearing agent
– Liquid in higher temperature and solid in room
temperature
– Homogenous and stable
– Non-toxic, odorless and cheap
– Transparent
– Fit for sectioning the tissue
– Capable of flattening after ribboning
– Easy to handle
– Inexpensive

34. • The time duration and the number of changes
required for the impregnation in tissue
depends on:
– Size of tissue: Thicker large tissue takes more time
to impregnate with the embedding medium. It
also contains more clearing agent to remove.
– Type of tissue: Hard tissue such as bone and
cartilage takes more time for embedding than soft
tissue.
– The type of clearing agent: Certain clearing
agents are easy to remove than others. Such as
xylene and toluene are easy to remove than

35. Different Impregnating Medium
• Paraffin Wax
• Dimethyl sulphoxide DMSO

36. Paraffin Wax
• Most popular universally accepted
impregnating and embedding medium for
tissue processing.
• Non-toxic and inexpensive medium
• Melting point of paraffin wax varies from 39 °C
-70 °C.
• In Indian subcontinent, the paraffin wax with
melting point around 60 °C is the most
suitable for laboratory use.
• Total 3–4 h’ time in paraffin wax is sufficient

37. Advantages:
• Easy cutting of serial sections
• very rapid processing
• Tissue blocks and unstained mounted sections
may be stored in paraffin for an indefinite period
of time after impregnation without considerable
tissue destruction.
• Because formalin-fixed, paraffin-embedded
tissues may be stored indefinitely at room
temperature, and nucleic acids (both DNA and
RNA) may be recovered from them decades after
fixation, they are an important resource for
historical studies in medicine.

38. Disadvantages
• Overheated paraffin makes the specimen brittle.
• Cause tissue shrinkage and hardening in case of
prolonged impregnation.
• Inadequate impregnation will promote retention
of the clearing agent. Tissues become soft and
shrunken, and tissue blocks crumble when
sectioned and break up when floated out in a
water bath.
• Paraffin wax takes long duration for the
impregnation of the bone and eye. otherwise,
they will crumble on sectioning.
• Paraffin processing is not recommended for fatty
tissues. Fat dissolves in dehydrating and clearing

39. • 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 stearic acid
• Reduction of melting point: addition of
phenanthrene
• Improving adhesiveness with tissue and wax:
addition of 0.5% of ceresin

40. Dimethyl sulphoxide DMSO
• The addition of small amount of DMSO in
paraffin wax
– reduces the infiltration time of the wax
– removes the residual clearing agent.
– It produces a homogenous matrix and better
support.

41. Embedding
• Embedding (Casting or Blocking) is the
process by which the impregnated tissue is
placed into a precisely arranged position in a
mold containing a supporting medium which
is then allowed to solidify.
• This supporting media for blocking known as
embedding media.
• Aims of embedding: Embedding medium has
three important functions:
– To give support of the tissue

42. • Done by:
– Filling mould of suitable size with molten
wax/embedding media
– Orientation of tissue in mould to ensure it will cut
in right plane
– Cooling of mass to promote solidification

43. • Different embedding medium:
– Paraffin wax
– Paraplast
– Carbowax
– Raisin
– Agar
– Gelatin
– Methacryate
– Celloidin

44. The choice of the embedding medium
• 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.
• Type of microtome
• Type of microscope

45. Paraplast
• Mixture of highly purified paraffin and
synthetic plastic polymers, with a melting
point of 56-57°C.
• More elastic and resilient than paraffin wax
thereby permitting large dense tissue blocks
such as bones and brain to be cut easily.
• Better ribboning of sections.
• Serial sections may be cut with ease, without
cooling the tissue block, thereby preventing
the formation of ice crystal artefact.
• Soluble in common clearing agents and

46. Carbowax
• a polyethylene glycol containing 18 or more
carbon atoms, which appears solid at room
temperature.
• It is soluble in and miscible with water; hence
does not require dehydration and clearing of
the tissue.
• The tissues are fixed, washed out and
transferred directly into the melted Carbowax.
• Processing time is reduced.

47. • For routine processing: Temperature of 56°C
are used
– I – 70% carbowax for 30 min.
– II – 90% carbowax for 45 min
– III – 100 % carbowax for 1 hr.
– IV - 100 % carbowax for 1 hr.
– Specimens are then embedded in fresh Carbowax
at 50°C and rapidly cooled in a refrigerator.

48. • Disadvantages:
– Carbowax is very easily dissolved in water. Hence
care must be taken to avoid contact of the block
with water or ice.
– Tissue sections are very difficult to float out and
mount
• Adding soap to water or using 10%
Polyethylene Glycol 900 in water will reduce
tissue distortion and promote flattening and

49. Epoxy resin
• Mainly used in electron microscopy as it
provides better resolution and greater details
of tissue.
Agar gel:
• helps in cohesion of friable and fragmented
tissue particularly in cytology sample and also
endometrial curetting and small endoscopic
biopsies.
• It does not provide good support of the tissue

50. Gelatin
• :It is also used in small friable tissues and
frozen section containing friable and necrotic
tissue.
• The melting point of gelatin is 35–40 °C, and
this low melting point makes it unsuitable for
embedding

51. Methacrylate
• Methacrylate monomer is miscible with
ethanol.
• In the presence of catalyst (benzoyl peroxide
2%),it is polymerized and provides a hard and
clear block.
• .

52. Celloidin
• Celloidin is purified form of nitrocellulose.
• Mainly used for specimens with:
– large hollow cavities which tend to collapse, f
– For hard and dense tissues such as bones and
teeth
– for large tissue sections of the whole embryo.
• Main disadvantages:
– Celloidin impregnation is very slow
– inability to cut thin sections.
– Serial sections are difficult to prepare

53. Different Types of Mould Used
for Block
• Leuckhard Embedding Moulds ( L moulds):
•

55. • The two L-shaped arms 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 of the tissue.
• The molten wax is poured in the space
between two L arms, and then 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

56. Stainless Still Mould
Plastic Mould

57. Tissue Embedding Method
• It is essentially same for all the types of
embedding media.
• The following things are needed for tissue
embedding:
– Molten paraffin wax
– Mould with cover
– Metal plate (cold plate)

58. Tissue tek system

60. Steps of Tissue tek system
• Liquid paraffin is kept at a constant
temperature in the dispenser of the system.
• The tissue is put on the lower surface of the
mould by forceps. The cutting surface should
be faced down.
• Molten paraffin is poured on the metallic
mould.
• The mould is covered with peripheral plastic
ring on the upper surface.

61. Tissue Orientation and Embedding

62. Thank you
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