Celloidin

Nitrocellulose, otherwise known as cellulose nitrate, goes Translate in

by several names. The commonest, in the histological Google Translate
world, are celloidin, collodion and parloidin, but others
Instructions
are not infrequently encountered, many of them ending in
"-oidin". Many of these names are well established trade
names for different companies' products.  
Cellulose nitrate
Nitrocellulose is also known as gun cotton from its use as
an explosive and its appearance, which can resemble cotton wool in the dry state and
can be produced from cotton, which is made of cellulose. As would be expected of an
explosive, it is extremely inflammable, a characteristic which is encreased when
dissolved in a mixture of absolute ethanol and ether, both either inflammable or
explosive fluids in their own right. Open flames, sparks from equipment, smoking
tobacco in any form and any other activity which can cause ignition of the material or
its solvent must be absolutely forbidden. Explosion safe processing and storage
containers are essential, and common sense must be used at all times. Fire
extinguishers, fire blankets and other fire safety equipment must be easily and
conveniently available at all times. Processing with nitrocellulose is safe if these
precautions are taken.

The celloidin which was originally used for histological processing produced a thick,
syrupy solution when dissolved in ethanol-ether (alcohol-ether). Later a material which
produced a less viscous solution for the same weight of nitrocellulose was introduced,
called low viscosity nitrocellulose (LVN). This made it possible to give greater support
to the tissues by using higher concentrations of nitrocellulose for the same viscosity.
Both products are still in use. As you can see from the formula, celloidin is a polymer of
nitrated cellulose units.

Celloidin embedding is a slow process, taking some weeks usually, and does not produce
sections as thin as those produced by paraffin embedding. The minimum thickness is
about 12 µ but 15 µ, or thicker, sections are often cut. Section cutting is usually done wet,
which means that the block is lubricated with a fluid, usually 60-70% ethanol, and is not
allowed to dry out. This makes section cutting somewhat messy and quite a bit slower
than the dry sectioning used with paraffin. When sections have been cut, they are then
stored in 60-70% ethanol instead of being adhered to glass slides. They are usually
stained free floating and put on slides at the same time as the coverslip is applied. This
does make it difficult to prepare serial sections as each section must be stored in an
individual, numbered container or small batches of 5 or so sections prepared. These are
not really "serial" sections but they are close. Blocks are stored in the same alcohol as
used for lubrication, and they must never be allowed to become dry.

Tissues embedded in celloidin are usually sectioned with a sliding microtome. In this
instrument the block is mounted on a platform facing upwards and is fixed. The knife is
held at a significant slant so that most of the blade edge is used during the cutting stroke,
and is quite long, often in excess of 25 cm. It is customary to use a deeply plano-concave
knife, the cutting stroke being to slice off a section rather than to peel it off, so that the
strength behind the knife edge is not needed but a very keen edge is necessary. The face
of the block is lubricated with 70% ethanol and the knife drawn across the top of the
block at a strong slant, shaving off a section, which is immediately removed and placed
in 70% ethanol. The surface of the block is then relubricated for the next cutting stroke.

The desirable aspects of celloidin processing are to do with it being a process that
completely avoids the use of heat at any stage. As a consequence, heat produced
artefacts are avoided. In particular, shrinkage is absolutely minimal, if there is any, and
organs composed of layers of materials of different characteristics do not have the
layers separate or shrink away from each other so the structural relationships of the
various types of tissue components can be seen clearly.

The undesirable aspects are the time it takes, the thickness of the sections, the necessity
for staining to be done on free floating sections with the celloidin still present, the
resulting restrictions on the variety of staining methods that may be used, the greater
difficulty in final dehydration and clearing which is caused by the absolute ethanol
softening the celloidin excessively, and the inconvenience of storing the blocks in sealed
jars with lids tight enough to stop evaporation of 70% ethanol completely.

The thickness of the sections is considered an advantage by some and a disadvantage by

others, depending on whether the microscopist wants to see multiple layers of cells and
the relationships between them as the microscope is focused up and down, or a single
layer without interference from surrounding material.

Making celloidin solutions

Celloidin is used in solution, usually in a 1:1 mixture of ethanol-ether at concentrations
of 2%, 4% and 8%. The equivalent concentrations for LVN are 5%, 10% and 20%.
Celloidin may be purchased either as a solution or as a solid, damped with a liquid to
reduce flammability. This is usually an alcohol, so its weight may be ignored when
dissolving. The stock purchased as a solution may be in an undesirable solvent and it is
often the practice to evaporate the solvent to obtain dry celloidin, which is then weighed
and redissolved in the appropriate solvent. The evaporation to dryness is done slowly at
room temperature, without additional heat and in an explosion safe environment as a
fire safety precaution.

The fastest way to dissolve celloidin is to soak it first in half the final volume of
anhydrous ethanol to soften it (50 mL for each 8 grams celloidin), with intermittent
mixing in a tightly stoppered container. The next day, an equal volume of diethyl ether
is added and intermittently mixed until an evenly consistent solution is obtained. The
2% and 4% solutions may then be made by simple dilution of the 8% solution with an
equal parts mixture of ethanol and diethyl ether. The solution should be transparent,
without undissolved material, and should be stored in a completely closed container
which is ether resistant. Glass is suitable.

Fixation
Infiltrating celloidin into tissues is a long, slow process. For that reason there is
absolutely no point in rushing other parts of the process. Fixation should be complete. If
formalin is used, a week or longer would not be too much, although smaller pieces of
tissue would only need a couple of days. Tissues should be refixed after trimming to
ensure fixation is complete. Other fixatives may also be used, but the overriding concern
should be that they be allowed to fix for long enough. If necessary, any aftertreatment
must be done thoroughly. Since celloidin processing is such a long process, any unfixed
or inadequately fixed tissues may result in serious deterioration of the tissues before the
process is complete.

Dehydration
Dehydration should also be thorough and complete, taking a few days with several
changes of ethanol. For delicate tissues, gradual dehydration is strongly recommended
to avoid distortion from removing the water too fast. Keep in mind that celloidin
processing causes almost no distortion of tissue layers and the gradual increasing of the
ethanol concentration is important in producing this distortion free appearance. It does
mean that poorly fixed material has a longer time to deteriorate from residual moisture
before the ethanol content increases sufficiently to stop it, and this makes thorough
fixation important.

Infiltration
No clearing agent is used with celloidin and following dehydration with absolute
ethanol, the tissue may be placed in ethanol-ether. Ether is a lipid solvent and will
remove much of it from the tissue. The tissues should then be placed into the thinnest of
the celloidin solutions (2%) and left in a tightly capped container for an appropriate
length of time. How long is appropriate? That depends on the tissue and could be a week
or months. The denser it is, the longer it must be left to infiltrate. Experience is the best
judge, but care should be taken not to be impatient because, if the infiltration time is too
short and the center of the block is not properly infiltrated, short of removing all of the
celloidin and repeating the process from the beginning, a procedure that would take
longer than the original processing, there is nothing that can be done. It is far better to
allow an adequate period of time for infiltration.

This process is then repeated with the medium and thickest solutions of celloidin.
Infiltration time is as long or longer than for the thinnest solution and, for the same
reasons, should not be rushed.

Casting and hardening

When infiltration is complete the block has to be cast and hardened. Paper boats do very
well for this, and have the advantage that they may be cut off if the paper does not peel
away easily. The instructions below for making paper boats are from The Microtomist's
Vade-Mecum.
 "To make paper trays proceed as follows. Take a piece of stout paper or thin
cardboard, of the shape of the annexed figure; thin
post-cards do very well indeed. Fold it along the
lines a a' and b b', then along c c' and d d', taking
care to fold always the same way.
 
Then make the folds A A', B B' , C C', D D', still
folding the same way. To do this you apply A c
against A a, and pinch out the line A A', and so on
for the remaining angles. This done, you have an
imperfect tray with dogs' ears at the angles. To
finish it, turn the dogs' ears round against the ends
of the box, turn down outside the projecting flaps
that remain, and pinch them down."

Some thick celloidin is poured into the bottom of a

boat, then the tissue arranged in it and more celloidin poured in to well cover the tissue,
keeping in mind that as the block hardens the celloidin will shrink. If at any time the
celloidin shrinks enough to expose the tissue, more celloidin should be poured in to
cover it. At the end of the hardening process there should be sufficient celloidin to allow
for trimming the back of the block flat so that it may act as a base for glueing the block
to a wooden holder for sectioning. Usually 5 mm or more is needed.

After filling the boat with thick celloidin it is placed under a bell jar, or something
similar, with a base that ensures air is excluded. A plate of glass is satisfactory. Each day
the top of the jar is lifted a little for a few minutes so that evaporated ethanol-ether can
escape. During the following day more solvent will evaporate from the block and the
atmosphere inside the bell jar will become saturated again. Evaporating the solvent off
slowly in this manner ensures that the celloidin thickens and hardens evenly
throughout the tissue. Evaporating it too fast will result in the outside of the block
becoming hard while the inside is still soft. Using this method of hardening the block
also allows the concentration of celloidin to increase and give additional support to the
tissue. The hardening process takes some time and is complete when the block is
sufficiently hard, often judged by pressing it with a finger nail and having some
difficulty leaving an impression.

At any time during the hardening procedure, it is possible to bring it to completion

within about 24 hours or so, but with somewhat less support to the tissue since the
celloidin will not have had an opportunity to properly concentrate. Simply place a small
open container of chloroform under the bell jar. The chloroform will saturate the
atmosphere and harden the celloidin without further evaporation.

Sectioning
Once hardened, the block is removed from the paper boat, preferably by peeling, but it
can be cut away with a sharp blade if necessary. The block is then trimmed, allowing
about 3 mm or so celloidin all around the tissue, then a distinctive cut is made on one
corner for orientation. The back is trimmed flat. The block is then placed into 70%
ethanol until ready to section.

Identifying the block with an accession number can be done by loading a sharp point
with India ink, then pricking a series of holes in the side of the block, leaving some India
ink in the hole. Extra ink may be rubbed over the holes to force some ink into them.
After the block has been sectioned a paper label may be attached to the side with
celloidin and covered.

To section, the block is first attached to a, usually wooden, holder. Place some thick
celloidin onto the holder and press the block into it, arranging it so that it will meet the
knife as wanted. A weight is then placed onto the block and the assembly is left until the
attachment is firm. The whole thing is then placed into 70% ethanol until ready for
sectioning.

Sectioning is best demonstrated and taught by an experienced technologist. It is done

slowly, the sections being sliced off the block individually and immediately placed into
70% ethanol. The block surface is wetted with 70% ethanol after each section is
removed, and it is never allowed to dry out. Roughing down is done in the same fashion,
and the urge to remove too thick a section must be resisted. In celloidin sectioning,
patience is an absolute necessity.

References
Wikipedia has an article about diethyl ether.
Bolles-Lee, A. (1913)
The Microtomist's Vade-Mecum Ed. 7.
P. Blakiston, Philadelphia, USA

 
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Last updated January 2019