1.
) Factors affecting ; (i) section thickness
(ii) section cutting
(iii) floatation
Section thickness
Embedding
Under-filling the cassette can cause unstable clamping in the microtome and lead to
cutting “thick then thin” sections.
Type of embedding compound.
The type of embedding compound reflects the thickness at which sections can be cut. It is
difficult to cut very thin sections from soft embedding compounds such as gelatin
compared to relatively harder embedding compounds such as paraffin and resin.
Microtome
The advance feed is set to the desired thickness of varying micrometers (3 to 5 µm for
most purposes).
In addition factors such as speed of rotation can also influence the actual thickness
achieved.
Microtome blade
That is the condition of the blade edge , how sharp or dull it is also if it has nicks etc. is a
major factor affecting section thickness.
Clearance angle
The clearance angle is the angle between the knife edge bevel and the block and is one of
the factors that can influence the actual thickness of a section.
The correct clearance angle is used to prevent compression in cut section and is also
important to reduce friction as the knife edge passes through the block.
Section cutting
Type of blade
Plane wedge : Is used for paraffin and frozen sections.
Planoconcave : Is used for celloidin sections since the blade is thin it will vibrate when used for
other harder materials.
Biconcave : It is recommended for paraffin section cutting on rocking and sledge type of
microtome.
Tool edge : This is used with a heavy microtome for cutting very hard tissues like undecalcified
bone.
Orientation of block
The trimmed block should be fitted into the block holder and orientated so the edge
offering least resistance meets the knife edge first to allow for good section cutting.
Blade debris
Debris adhering to upper or lower edges of the block, or the back of the blade, can make
it difficult to obtain cohesive ribbons and cause the ribbon to lift off the blade on the
upstroke.
Cutting angle
This is the angle of bevel. If it is too great it can cause compression in the cut section. If
the angle is too fine the edge of the knife can vibrate causing chatter in the section. A
balance between these extremes will provide the best results.
In addition the sharper knife will have a finer cutting angle.
Microtome blade
That is the condition of the blade edge , how sharp or dull it is also if it has nicks etc. is a
major factor affecting section thickness.
Microtome settings
Making sure that all of the clamping mechanisms are tightened securely is a factor, as if
any are loose or not secure uneven sections can results, thick then thin sections and
unnecessary damage to the tissue block or microtome can occur.
Temperature of the block
Cold wax provides better support for the harder elements in a specimen allowing thinner
sections to be obtained.
Speed of rotation
Generally a slow, uniform cutting stroke produces the best results and the least
compression.
Floatation
Temperature
The temperature will need to be 5 - 9 ˚C below the melting point of the wax.
Water-bath
Make sure the water is clean and free of bubbles and section waste (to avoid cross-
contamination).
Duration
Leave the section on the water surface just long enough for it to flatten. Over expansion
can spoil the morphology in susceptible sections.
2.) Common faults encountered from embedding to placing sections on a slide.
Tissue embedding
Embedding is the process by which tissues are surrounded by a medium such as agar, gelatin, or
wax which when solidified will provide sufficient external support during sectioning.
Infiltration (Interpenetration)
Is the saturation of tissue cavities and cells by a supporting substance which is generally, but not
always, the medium in which they are finally embedded. Tissues are infiltrated by immersion in
a substance such as a wax, which is fluid when hot and solid when cold.
Paraffin wax permeates the tissue in liquid form and solidifies rapidly when cooled. The tissue is
impregnated with the medium, forming a matrix and preventing distortion of the tissue structure
during microtomy. It has a wide range of melting points, which is important for use in the
different climatic regions of the world. To promote desirable ribboning during microtomy,
paraffin wax of suitable hardness at room temperature should be chosen. Heating the paraffin
wax to a high temperature alters the properties of the wax.
Embedding involves the enclosing of properly processed, correctly oriented specimens in a
support medium that provides external support during microtomy. The embedding media must
fill the matrix within the tissue, supporting cellular components.Most laboratories use modular
embedding centers, consisting of a paraffin dispenser, a cold plate, and a heated storage area for
molds and tissue cassettes.
Paraffin wax is dispensed automatically from a nozzle into a suitably sized mold. The tissue is
oriented in the mold; a cassette is attached, producing a flat block face with parallel sides. The
mold is placed on a small cooling area to allow the paraffin wax to solidify. The quick cooling of
the wax ensures a small crystalline structure, producing fewer artifacts when sectioning the
tissue.
Orientation of tissues
Specimen orientation during embedding is important for the demonstration of proper
morphology.
Incorrect orientation may result in diagnostic tissue elements being damaged during microscopy
or
not being evident for pathology review. Products are available that help ensure proper
orientation:
marking systems, tattoo dyes, biopsy bags, sponges, and papers. Orientation of the tissue should
offer the least resistance of the tissue against the knife during sectioning.
Avoid under-filling the cassette as this can allow unstable clamping in the
microtome and lead to cutting “thick then thin” sections and other problems.
• Avoid over-filling cassettes as this can interfere with the correct alignment of the
block face for sectioning.
• Any excess wax on the outside of a cassette should be removed before clamping to
ensure the block is firmly held during sectioning.
Microtomy
Microtomy is the means by which tissue is sectioned and attached to a surface for further
microscopic examination. Most microtomy is performed on paraffin wax embedded tissue
blocks. The basic instrument used in microtomy is the microtome; an advancing mechanism
moves the object (paraffinblock) for a predetermined distance until it is in contact with the
cutting tool (knife or blade). The specimen moves vertically past the cutting surface and a tissue
section is produced.
Microtome knives and disposable blades are extremely sharp and can inflict
serious injuries unless appropriate care is taken when working with them.
Accidents occur when a microtomist is distracted and not concentrating fully.
• Use forceps or brush instead of your fingers to pick up sections or wax fragments
from blade or block face.
The handwheel lock will lock the object head at the top of the cutting stroke and
must be used when changing blocks.
• The guard must be in place and the handwheel lock engaged when a block is being
placed into or removed from the cassette clamp, or when any manipulation of the
block is being undertaken while the knife or blade is in place. The guard must also
be used when the microtome is left unattended.
• The handwheel brake will lock the microtome when the handle is in any position
and is used when realigning a block face or adjusting the coarse feed.
Sectioning is generally improved when the specimen and the wax are well matched in
hardness. It is for this reason that most paraffin blocks must be cold when sections are
cut. The actual method used to chill the block is important.
• Cold wax provides better support for the harder elements in a specimen allowing
thinner sections to be obtained.
• Place the blocks on a cold plate or a cold wet surface for a few minutes (such as
the surface of melting ice).
• Water penetrates a small distance into the block face, swelling tissues and making
them more amenable to cutting. This is particularly important to over-dehydrated,
dry or crumbly tissues.
Cutting thick and thin sections
Thick
Wrong micrometer setting
• Warm breath applied to cold block
to facilitate sectioning
• First section in ribbon chosen
• Sectioning at too great a speed
• Poor processing
• Microtome needs recalibration
Holes
• Block trimmed too quickly
• Block surface not polished by
cutting some thin sections after
roughing
• Inappropriate section thickness
used when trimming
• Block brittle (over-processed?) or
too cold when trimmed
Microtome settings
Making sure that all of the clamping mechanisms are tightened securely is a factor, as if
any are loose or not secure uneven sections can results, thick then thin sections and
unnecessary damage to the tissue block or microtome can occur. Since a loose knife or
loose block can cause this.
Microtome
The block might be too large for the microtome in use. If there is very little clearance
between the bottom of the block and the top of the knife it can be difficult to cut through
tissue with a smooth action. This often results in thick and thin areas in the resultant
section.
Clearance angle
If the knife clearance angle is too fine this produces an effect at the knife edge similar to
a blunt knife. The block face is much too close to the bevel so that as the knife cuts, there
is a small expansion of the block face caused by the friction between the two surfaces
forcing the block face up against the bevel making it very difficult to cut in a smooth
action and usually producing thick and thin areas in the section.
Areas of calcification present in tissue can cause the knife to jump and vibrate and so can
produce thick and thin areas in the section as well as damaging your knife edge. If minute
areas of calcification are present surface decal for 15 to 30 minutes should remove the
offending particles.
Ribbons split vertically or scratch lines appear
small nicks in the knife edge which can be caused by handling the knife badly
Clearance angle
If the knife clearance angle is too great a build up of debris can occur on the knife edge, and that
debris can cause scratch lines in sections rather than the clearance angle itself causing scratch
lines to appear.
Areas of calcification present in tissue can cause the knife to jump and vibrate and so can produce
thick and thin areas in the section as well as damaging your knife edge. If minute areas of
calcification are present surface decal for 15 to 30 minutes should remove the offending particles.
Sections adhere to the block on the upstroke of the microtome
Clearance angle
A knife clearance angle too fine causes the block to rub against the knife back usually
causing deposition of wax on the back of the knife which in turn causes sections to stick
to the knife.
Condition of blade
If the knife is blunt then sections will compress and compressed sections tend to stick
together and bunch up on the knife edge. So both of the above, can and probably will,
produce adhere.
Sections roll up when cut
Condition of blade
A blunt knife can cause this to happen as well as if the Knife clearance angle is too great
because having the angle too great is very much like presenting a blunt edge to the block.
Microtome setting
If the micrometer is set too thick then sections can also roll up. Ideally sections should be
cut at 3 to 5µ to demonstrate most tissue elements, but if you must have thick sections
like those that are required to demonstrate some neurological elements then make sure
that your knife is very sharp.
Temperature of block
If the block is too warm this can also cause the sections to curl.
Sections will not ribbon
Clearance angle
A knife clearance angle too fine or too great can result in sections not ribboning. Both we
have discussed so there’s no need to go into it again. If your knife clearance angle is
always correct then all of the problems associated with incorrect clearance angles just
will not happen.
Condition of blade
A dirty knife edge usually causes sections to stick to the knife edge so that it is difficult to
get them to ribbon. So it is important to keep the knife edge clean, especially the back of
the knife. Use xylene followed by alcohol. If you use xylene alone being an aromatic
hydrocarbon and slightly oily it does leave a fine film of material on the back of the knife
ready to catch the next section. Alcohol removes the oily film and leaves the surface very
clean.
Sections crumble or tear
Condition of blade
A blunt knife will cause sections to crumble,
Temperature of block
if the Wax too soft sections can also disintegrate.
Crooked or uneven ribbons when sectioning
If the ribbon is straight you will get more sections on a slide, like with tiny biopsies where you
may put several sections on the one slide. It is easier for the pathologist to move from section to
section down the slide.
Microtome settings
the edges of the knife and block are not parallel, if the top and bottom are not parallel
then your ribbon will bend.
and not straight or the knife is not straight in the knife holder.
Wrinkled or compressed sections
Condition of blade
a Blunt knife. Blunt knives do cause many problems and if your knife is blunt the section
will wrinkle because of the different densities between the wax and tissue and the
different densities of the components within the tissue. The different densities offer a
range of conflicting forces at the knife edge all of which cut at different rates if the knife
is blunt causing those dreaded wrinkles.
Temperature of block
a warm block
A harder, colder material can be cut with less friction than soft, warm material if the
knife is sharp. The softer, warmer material will wrinkle more easily.
Clearance angle
If a Clearance angle is too great there is far more friction as the block meets the edge of
the knife which causes some compression and wrinkling.
Secure/stability
a Loose knife can cause all sorts of problems, wrinkling and compression being only two
of them. The most serious problem with a loose knife would be the blade digging deeply
into the block destroying the block face. If your lucky the tissue will not be destroyed in
the process and can be re-blocked. If your not lucky you may destroy the architecture of
the tissue. So make sure that your knife is held firmly in place.
Cutting angle
If the Bevel of knife is too deep making the cutting angle too shallow the effect is the
same as having a clearance angle too great. There is far more friction as the block meets
the edge of the knife which causes some compression. This is only a problem if you are
still re-sharpening the old style solid knives. It is not a problem with disposables.
And the Micrometer set too thin for hardness of wax. What usually happens when you try
to cut a section that is too thin for the wax you are using is that the section crumbles. But
wrinkling is also one of the symptoms of having the micrometer set too thin for the wax
type being used.
Floatation and Slide
Wrinkles appearing in sections floated on a flotation bath. There’s nothing worse than
cutting a great, flat, smooth section and then floating it onto the water bath and finding it
full of wrinkles.
This can be caused by the Flotation bath being too cold. If the water is too cold the wax
surrounding the section will not expand and flatten. And it is the expansion and flattening
of the wax which also flattens the section, if it is properly infiltrated with wax. Ideally the
flotation bath should be kept at temperature about 5°C below the melting point of the wax
being used.
Condition of blade
Or the Knife is too blunt (especially if wrinkles are in the vertical plane)
Sections disintegrate on the water bath
This can happen when the water temperature is too high. It has a tendency to soften and
melt the wax and once the wax breaks up so does the section.
If the wax is contaminated with clearing agent or if the block has not been properly
dehydrated then the section will also disintegrate. We discussed this one also when we
talked about the problems associated with sections crumbling.
There is no substitute for experience but there are some fundamental steps that will
make the task easier.
Use a section of blade that has not been used for rough trimming.
• When using the coarse feed, avoid cutting unintentional thick sections as this will
damage your knife and possibly the block face.
• Re-chilling of the block may be required if the block face becomes warm or if
deeper levels are required.
• Generally a slow, uniform cutting stroke produces the best results and the least
compression.
• Do not stop and restart during a cutting stroke as this will produce bands of
different thickness across the section.
• The practice of gently breathing on the face of a chilled block immediately before
cutting each section, is common practice in some laboratories. The application
of warm, moist breath tends to make sections more cohesive, but it also causes
thermal expansion thus making the section thicker3
• Debris adhering to upper or lower edges of the block, or the back of the blade, can
make it difficult to obtain cohesive ribbons and cause the ribbon to lift off the blade
on the upstroke. If debris is present clear it away, re-chill your block and start
again.
References
http://issuu.com/leicamicrosystems/docs/microtomy_and_paraffin_section_preparation/20
http://www.rmsc.nic.in/RHSDP%20Training%20Modules/Introduction%20(1).pdf
http://www.ihcworld.com/royellis/problems/mainpage.htm
