Chapter XXIV: The Preparation of Mounted Sections

Sections of fresh material, unimbedded or imbedded tissues must be treated by a series of processes before they are finally permanently mounted and ready for use. These processes in general are: =preparation for staining=, =staining=, =differentiation=, =washing=, =dehydration=, =clearing= and =mounting=. The general procedure will be modified to some extent by the character of the tissue, manner of preparation (fixed or unfixed, imbedded or unimbedded, unstained or stained), character of stain (affected by alcohol, xylol, etc.), and the mounting agent (glycerin, balsam, damar, colophonium). Two or more of these steps may be combined in one; the same agent may differentiate, dehydrate and clear. Several stains may be combined in one solution, or it may be necessary to use them in succession. The very greatest variation is possible in pathologic technique; in fact, practically every laboratory worker modifies methods according to the light of his individual experience. The really important thing is to be master of the method, and not allow the method to control the situation. One of the greatest attractions about laboratory work is the infinite possibility of variation and improvement of methods and the invention of new ones.

I. PREPARATION FOR STAINING.

a. =Frozen Sections of Fresh Tissue.= Frozen sections of fresh
tissues, as well as those obtained by the single or double razor,
may be stained by floating the section on a slide, staining it
directly (carbol-kresyl-echt-violett or carbol-thionin), examining
in the stain or washing, dehydrating, clearing and mounting; or the
section may be fixed to the slide with molasses or sugar-dextrin
solution, covered with a celloidin-film, and treated according to
the methods followed for paraffin or celloidin sections. Sections
of fresh tissue may be fixed in formol or alcohol, and then treated
by the same methods as celloidin sections. (See also Page 220.)

b. =Sections of Unimbedded Tissues.= These may be handled for
staining in the same way as paraffin, celloidin or fresh-tissue
sections, either when sectioned directly or after freezing. The
sections may be stained directly, on the slide, cover-slip, or in
the staining solution, or they may be transferred into celloidin
sheets by the same methods employed in the preparation of paraffin
sections.

c. =Celloidin Sections.= These may be transferred from water or
alcohol directly to the stain. It is not necessary to remove the
celloidin. If not stained soon after cutting they should be
preserved in 95 per cent alcohol. Celloidin sections may be stained
on the slide by simply blotting the section firmly on the slide,
without permitting it to become dry, and manipulating it carefully
through the various solutions; or the section may be fixed to the
slide by the use of 95 per cent alcohol, ether-vapor and fixation
in 80 per cent alcohol; or the section may be fixed to the slide
by the methods given above under the cutting of serial sections
of celloidin blocks. The most common method of preparation of
celloidin sections for staining is to transfer the sections from
alcohol into water to straighten them out, and then to transfer on
the spatula into the stain. For the treatment of serial celloidin
sections see above.

d. =Paraffin Sections.= Paraffin sections may be stained directly
_without removing the paraffin_. This is especially advisable in
the staining of tubercle-bacilli and in other cases where the use
of alcohol is to be avoided. For many stains this method cannot be
used. The sections as they are cut are floated directly into the
warm stain, on which they flatten out, and are then transferred to
the other solutions on the section-lifter, finally dried on the
slide, in the incubator or over the flame, cleared in xylol and
mounted in balsam. Paraffin sections may also be stained without
removing the paraffin by being transferred directly from the
knife on to 80 per cent alcohol, stained, washed, dehydrated in
absolute alcohol or by drying, cleared and mounted. The section is
transferred from one solution to another on the slide or spatula.
The paraffin is removed during the clearing in xylol in both of
these methods. The treatment with xylol must be on the slide, else
the section may go to pieces. The staining of the section in the
paraffin usually takes more time than staining after the paraffin
has been removed, but the process can be hastened by heating the
stain.

_Slide and Cover-slip Preparations._ Paraffin sections may be
affixed to a slide smeared with a thin film of albumin-glycerin
(equal parts of filtered beaten white of egg and glycerin, with
crystal of phenol or thymol, or 1 grm. of sodium salicylate to
100 grms. of the mixture as a preservative). A drop of fixative
is placed upon a clean slide, and is rubbed over the slide in a
fine film with the back of the finger. The dry paraffin section
with glossy side down is placed upon the smeared slide, flattened
with a brush and then pressed firmly against the slide with the
ball of the thumb. The albumin-fixative is then coagulated in
the incubator or over the flame; the paraffin is melted over the
flame without over-heating the section and the slide covered at
once with xylol to remove the paraffin. It is then put into 95
per cent alcohol, thence into the stain; and after staining, the
section is washed, dehydrated, cleared and mounted. _Cover-glass_
preparations of paraffin sections are made by floating the
sections with glossy side downward on warm water (just below the
melting-point of the paraffin) until they straighten out and are
perfectly flat. They are then floated on to cover-slips covered
with a thin film of albumin-glycerin, the albumin having previously
been coagulated by passing the smeared cover-slips through the
flame quickly so that they do not scorch or burn. The cover-slips
with the adherent sections are then placed in the incubator for 12
hours. The paraffin is then removed by xylol, the xylol is washed
out in 95 per cent alcohol, and the cover-slips are then carried
through the processes of staining, washing, dehydration, clearing
and mounting. The cover-slips must be handled with forceps and
the section side should always be uppermost. Slides covered with
a film of albumin-glycerin may be used instead of cover-slips.
The albumin-glycerin film may be omitted, and the sections with
glossy side down floated in warm water on to clean covers or
slides; the water is drained off and the slides or covers are put
in the incubator for 12 hours. Sections adhere fairly well by this
method (_capillary attraction method_). Bubbles are removed by
careful heating. Serial ribbons of the size desired can be floated
and mounted on slides by the albumin-glycerin or the capillary
attraction method.

By far the best method of preparing paraffin sections for staining
is the _molasses plate method_, a modification, originating in
my laboratory, of the _Schmorl-Obregia_ sugar-dextrin method.
When many sections are to be stained at once it is the most
convenient method and gives uniform results. In the preparation
of sections for class-work it has no equal. It can be used also
for giving out unstained sections. When many sections must be
stained in diagnostic work the method saves much time and labor.
Fifty sections can be stained as easily as one. It combines all
the advantages of the celloidin and paraffin methods, as does the
_Schmorl-Obregia_ sugar-dextrin method, but is much cheaper than
the latter.

_Schmorl_ advised the use of a sugar-dextrin solution (cane sugar
solution [1:1] 300 cc., 80 per cent alcohol 200 cc., yellow
dextrin solution [1:1] 100 cc.) to be run over a perfectly clean
glass plate or slide until the entire surface is covered with an
even layer. The paraffin sections as they are cut are arranged in
order on the wet plate, and when the plate is full, it is heated
sufficiently to flatten and smooth the sections. The plate is then
placed in an incubator for 3-12 hours to harden and dry. When dry
it is immersed in xylol to take out the paraffin, then treated
with absolute alcohol for 10-15 minutes, the alcohol drained off,
and the plate covered with a thin layer of celloidin (celloidin or
photoxylin 10, absolute alcohol 100, ether 100). As soon as the
celloidin sets (1-2 minutes) the plate is immersed in warm water
and the celloidin film containing the sections is detached. It
can now be carried through the staining, washing, dehydrating and
clearing solutions as one section, and in the clearing solution cut
into strips or single sections, as desired, for mounting. _Huber
and Snow_ improved the method greatly by floating the paraffin
sections directly on to warm dilute sugar-dextrin (a 10 per cent
solution of Schmorl’s stock-solution will suffice), and plating
the sections directly from the latter. This method of using the
dilute solution is less expensive, much cleaner, and saves time
in drying in the incubator. The results are in every way better
than with the Schmorl solution in full strength. The formation of
bubbles and crystals is almost wholly prevented, and less dust is
caught on the plate. In my laboratory we have modified the method
still further by using a 10 per cent solution of New Orleans black
(or baking) molasses instead of the more expensive sugar-dextrin
solution. As the molasses costs but 20 cents a gallon, a gallon of
the dilute solution costing 2 cents can be used indefinitely if
fermentation be prevented by a crystal of phenol or thymol. The
paraffin sections are floated on to this dilute molasses solution
warmed sufficiently to smooth out the sections; 4 × 5 glass plates
(old negatives) thoroughly cleaned and kept in alcohol are immersed
in the warm molasses solution and the sections arranged on them as
desired, lifting out of the solution that part of the plate covered
with sections as they are drawn upon it. As soon as the plate is
covered it is drained, and is then flooded with absolute alcohol.
After 1-2 minutes the alcohol is drained off and the plate flooded
with thin celloidin, which is allowed to set for a minute or so,
and the plate then immersed in warm water in which the celloidin
film containing the paraffin sections is detached. This film is
then handled by catching it at the two corners of one end with
the fingers, or better still by a pair of forceps held in each
hand. The film is put first into xylol to remove the paraffin,
then into 95 per cent alcohol, then into water and thence into the
staining solution. After staining the film is washed, dehydrated
and cleared, and in the clearing solution is cut into strips or
single sections by means of the wheel-shaped paper-cutter used by
paper-hangers. The pieces are then mounted. A dilute sugar-dextrin
solution can be used instead of the molasses-solution, but the
latter is much cheaper and does just as well. Aside from this
advantage our method of transferring the paraffin-sections into the
celloidin film without first removing the paraffin saves a great
deal of time, as it is not necessary to wait for the plates to dry
in the incubator. The same method can be applied to the staining
of single paraffin sections on the slide. The conversion of the
paraffin section into a celloidin preparation without any loss of
time for drying is so quickly and easily carried out that I advise
it above all others. The same method may also be applied to the
staining of fresh and fixed tissues cut on the freezing microtome
or sectioned without imbedding. The success of the plate-method
will depend largely upon the state of the glass-plates when put
into the molasses solution. They must be perfectly clean or the
celloidin sheet will not separate well. It is best to keep them
in alcohol until they are needed. The celloidin must be of the
right consistency, the layer must be thin, and cover the entire
plate uniformly. It must not be allowed to harden too much before
immersion in water or it will be tough and will shrink. Handling
of the celloidin-sheets with the bare hands is not advisable
because of the large number of epithelial cells adhering to the
celloidin. The sheets are easily changed from one solution to
another by catching them with forceps; the use of a glass-plate
to transfer them is not necessary. When it is desired to preserve
sections for future staining the celloidin sheet containing the
paraffin-sections can be kept in 80 per cent alcohol indefinitely.

NOTE:—If the glass-plate is numbered with a blue wax-pencil
after the paraffin sections are floated on, the marking will be
transferred to the celloidin sheet, and the latter will retain the
marking through all solutions.

II. STAINING AND DIFFERENTIATION.

Staining is necessary to bring out clearly the constituent elements of the tissues and their relations with each other, and for the demonstration of histologic structures or chemical substances that would otherwise be nearly or wholly invisible. The technique of staining depends upon the fact that stains or dyes possess certain affinities for the tissue-elements or for certain simple or complex substances present in the tissues (_microchemic reactions_). These affinities vary greatly with the dye. Some dyes have an affinity only for single constituents of the tissue (_elective_ or _specific stains_); others have an especial affinity for the nucleus (_nuclear stains_), others stain all the tissue-constituents diffusely (_diffuse_ or _protoplasmic stains_). There are but few pure elective stains for single tissue-elements; the majority of stains will stain more than one of the tissue elements, but may show an especial affinity for certain ones. As a result of these variations in the affinities of dyes for the constituents of the tissues it becomes possible to manipulate the dyes or to combine them in such a way that a specific differentiation of many tissue-elements is possible through the use of _different methods of staining_. These methods are based in part upon the use of different mordants, the employment of several stains in combination or in succession, the mixture of stains to form a new staining compound, the phenomenon of _metachromasia_, the differentiation of certain tissue-elements by the removal of the stain from the structures for which it possesses a weaker affinity, and by the employment of different microchemic reactions. The two most commonly employed methods are the _progressive_, in which the stain is allowed to act until the affinities of certain tissue-elements have been satisfied when the staining process is interrupted; and the _regressive_, in which the tissue is over-stained, and the dye withdrawn from the tissue-elements for which it possesses the weakest affinities leaving the other elements stained. This latter process is usually called “_differentiation_,” and the chief substances used for such differentiating are dilute acid, acid alcohol, acid stains, aniline oil, aniline-xylol and alcohol. Some workers use the regressive method for such simple stains as hæmatoxylin, overstaining, and then differentiating with acid alcohol before counterstaining with eosin. The results obtained in this way are much less satisfactory than is possible with the progressive method.

Tissues may be stained in the body during life (_intravital staining_), or immediately after removal from the body (_supravital or survival staining_), either before or after sectioning. (See Page 217.) Fixed tissues may be stained in bulk or in sections.

Staining Tissues in Bulk.

This method is not often used in pathologic work. The fixed and
hardened tissue is cut into small pieces, placed in the staining
solution for several days, washed thoroughly, dehydrated in
alcohol, imbedded, cut, and mounted without further staining.
Alcoholic solutions penetrate best; hæmatoxylin, hæmalum, carmine
and alcoholic solutions of the aniline stains may be used.
_Metallic impregnation_ (gold or silver salts) of fresh or fixed
tissues is but little used in pathology. (See Staining of Nervous
System, and Spirochætes.)

Staining of Sections.

=Celloidin sections= are lifted from water or alcohol into the
stain by the needle or section-lifter. The use of the latter
is advised, as by it the section can be floated flat on to the
staining solution. When many celloidin sections are to be stained
at once they can be stained in small tea-strainers and transferred
in these from one solution to another. =Paraffin sections= may be
floated directly on to the stain without removing the paraffin; or
they may be stained on the slide or cover-glass after removing the
paraffin, the stain being dropped on to the section, or the slide
or cover-slip is immersed in the stain. Special staining-dishes
for the staining of paraffin sections on slides and covers can be
obtained. Paraffin sections transferred to celloidin sheets by the
plate method can be put into the staining-solution while on the
glass-plate, or the films can be detached and transferred from one
solution to another by means of forceps. This is the easier way,
and it is not necessary to touch the films with the fingers.

General Rules for Staining.

1. The stain should be filtered just before being used, in order
to remove precipitates, moulds, etc. Unless they have been diluted
most of them can be used over and over again, hence after using
they should be filtered back into the stock bottle.

2. A liberal amount of stain should be used. Slides and cover-slips
are given enough stain to cover completely the section, when
the staining is done on the slide, or they may be immersed in
staining-dishes. Plates and celloidin sheets should be stained in
large trays. Sections and celloidin films should be flat without
folds or wrinkles, and they should not touch one another when
several are stained at the same time. Transference of the section
from water or dilute alcoholic solutions to dilute or stronger
alcohol respectively for a moment and then back again will usually
straighten out curled or wrinkled celloidin sections.

3. Stain until the section is properly stained. Control this by
removing it from the staining-solution and examining it in water on
a glass-slide without a cover-slip, using the low-power. Sections
will always appear more deeply-stained when cleared than when
examined in water, hence due allowance should be made. Celloidin
sheets can be examined on glass-plates. The time-limits given in
staining methods are only approximate; no absolute rules can be
laid down as to the length of time necessary to obtain a good
stain. The methods of fixation and hardening, age of the tissue,
age of the stain, etc., affect the staining power. Some stains
lose their staining-power after a time; others require a period of
ripening before they yield the best results. As a rule staining
may be intensified or hastened by staining in the incubator or at
a higher temperature, by concentrating the stain, or by the use of
such substances as aniline oil. When differentiation is necessary
the process should also be controlled by frequent examination of
the section, as above for staining. Usually the section can be
examined in the differentiating fluid.

III. WASHING.

Thorough washing after staining is necessary after nearly all
stains. The washing should usually be done by soaking the sections
in several changes of distilled water, although tap-water, alcohol,
alum-water, and other solutions may be used to intensify the
staining effects. When this is done a final washing in distilled
water or alcohol is usually necessary. Differentiating fluids
should always be thoroughly removed from the section before
mounting. Sections should not be allowed to lie in wash-water that
is colored by the stain; as soon as the wash-water becomes colored
it should be replaced by fresh. When sections are left lying in
the wash-water for some time the vessel containing them should be
covered to prevent the settling of dust on the sections, as it is
practically impossible to remove from the latter dust or other
precipitates that may become attached to them. Some stains give
better results after long washing; others are easily washed out if
the sections are left standing in the wash-water. The time-limits
of washing will depend upon the character of the stain employed.

IV. DEHYDRATION.

Dehydration of the sections is usually produced by passing them
through two alcohols, 80 per cent and absolute, or 80 per cent and
95 per cent. For certain clearing reagents (xylol) it is necessary
to use absolute alcohol. When carbol-xylol is used as a clearing
reagent absolute alcohol is not necessary, and 95 per cent can
be used instead for the second dehydrating solution. Usually a
minute in each alcohol is sufficient for the dehydration of single
sections. If dehydration with alcohol is not desirable because of
its action on the stain it is possible to dehydrate and clear in
xylol by repeatedly blotting the section with absorbent paper,
covering the section several times with xylol and then blotting.
The section should never be allowed to become perfectly dry.
Dehydration with alcohol may also be avoided by staining paraffin
sections without removing the paraffin, drying in the incubator
or over the flame, removing the paraffin in xylol, and mounting.
Imperfect dehydration is shown by the presence of white spots or a
milky cloud in the section when it is put into the clearing fluid.

V. CLEARING.

After dehydration, sections must be cleared in some solvent of
balsam before they can be mounted in the latter medium. When 95
per cent alcohol has been used for the final dehydration the
sections may be completely dehydrated and cleared at the same
time by the use of carbol-xylol (xylol, 3 parts; melted crystals
of carbolic acid, 1 part; add melted carbolic acid to the xylol
to prevent formation of crystals). The sections (on the slide
or cover-slips, in celloidin sections or films) are transferred
from the alcohol, draining or blotting off excess of the latter,
into the carbol-xylol, and left until perfectly clear. This can
be most easily determined by viewing the sections against a dark
background. Carbol-xylol cannot be used for sections treated with
aniline stains. These are dehydrated in 95 per cent alcohol, and
the final dehydration and clearing accomplished by repeatedly
placing xylol upon the slide and blotting it out until the sections
are transparent. Turpentine, chloroform, benzine, toluol, the
oils of bergamot, cloves, thyme, lavender, origanum cretici, and
cedarwood, aniline oil, and various mixtures of these oils are
also used as clearing agents. The majority of these will clear
from 95 per cent alcohol, but not so readily as carbol-xylol; they
have individual disadvantages of taking out the eosin, affecting
aniline colors, dissolving celloidin, making sections brittle,
slow action, clinging odor, etc. Chloroform and benzine may be
used for clearing osmic-acid preparations; oil of turpentine is
also good for clearing sections stained with kresyl-echt-violett,
and Wright’s blood-slain. With but few exceptions carbol-xylol
and xylol meet all requirements better than any other clearing
reagents. There is but one disadvantage in the case of
carbol-xylol; some of the phenols in the market cause a fading of
eosin and hæmatoxylin stains. _DeWitt_ has shown that this fault
can be corrected by redistillation, stopping the distillation as
soon as the temperature begins to rise above the constant boiling
point of the phenol; or the carbol-xylol that fades the stains can
also be corrected by supersaturating it with a mixture of sodium
bicarbonate one part, and sodium-potassium tartrate two parts.
Sections kept in xylol or carbol-xylol should be protected from
dust and evaporation; it is not a good plan to keep them in these
solutions for more than 24 hours.

VI. MOUNTING.

Permanent mounts are made in glycerin, potassium acetate, lævulose,
glycerin gelatin, balsam, damar or colophonium. For celloidin and
paraffin sections a solution of =Canada balsam= in xylol is most
commonly used for mounting. Celloidin sections (celloidin films are
best cut into strips and single sections when in the carbol-xylol;
the wheel-shaped paper-cutter used by paper-hangers is the best
instrument for this purpose) are lifted onto the slide from the
clearing-fluid; folds or wrinkles in the celloidin are straightened
or removed by cutting the celloidin at right angles to the section
in order to relieve the tension, and the section is then blotted
firmly against the slide by means of a pad of absorbent paper.
The greatest care should be taken to prevent wrinkling, folding
or turning over of the edge of the section. As soon as the pad
is removed a drop of balsam is placed upon the section and the
cover-glass put over it. There should be just enough balsam used to
fill the space between cover-slip and slide, so that air-bubbles
are not formed. The balsam must not be so thin that the cover-glass
will float about on the liquid, or so thick that it does not spread
well. In the latter case warming the slide may cause it to spread
more readily, but care must be taken not to injure the stain by
over-heating. Paraffin sections on the slide are similarly blotted
and covered with balsam and cover-glass; those on cover-slips are
blotted between folds of absorbent paper and immediately placed
with section-side downward upon a drop of balsam that has been put
upon the slide.

=Xylol-damar= may be used in place of Canada balsam; it is cheaper
and colorless, but it tends to become cloudy. =Colophonium= is
the cheapest of the three and has but little color; it is highly
recommended by many workers. In a xylol-solution it may be used for
aniline stains; a chloroform solution is advisable for the mounting
of osmic-acid preparations; while a solution in turpentine and
shellac is recommended for Weigert’s neuroglia method, Wright’s
blood-stain, and other special staining methods.

=Glycerin=, =potassium acetate=, =laevulose= and =glycerin-gelatin=
are used for the preservation of amyloid-, mucin- and fat-stains,
as well as for other preparations that do not permit the use of
alcohol and xylol. =Glycerin-gelatin= is probably the best medium
for this purpose. It is made according to _Kaiser_ by soaking 7
grms. of gelatin for 2 hours in 42 cc. distilled water, then adding
50 grms. glycerin and 1 grm. carbolic acid; the mixture is warmed
10-15 minutes, stirring constantly, and filtered while hot. It is
also made by taking water, 200 cc.; gelatin, 20 grms.; powdered
white shellac, 2 grms.; Farrant’s solution (gum arabic, glycerin,
solutio acidi arsenicosi conc., _aa._ 30.0 grms.), dissolve by
warming, and filter while warm. To mount in this medium the section
is placed on the slide, and blotted with absorbent paper. A drop
of warm glycerin-gelatin is then placed on it and the cover-slip
affixed. The drop spreads evenly beneath the cover-glass and
becomes solid as it cools. Mounts in glycerin, glycerin-gelatin,
potassium acetate and laevulose must be cemented around the borders
of the cover-slip with asphalt, wax, paraffin, gold size, etc.,
using a brush or glass rod for this purpose.

VII. SUMMARY OF METHODS OF PREPARATION OF CELLOIDIN SECTIONS.

1. Fixation of the tissues in alcohol, formol, etc.
2. Wash 24 hours, when necessary.
3. After-harden in 80 and 95 per cent alcohols for 1 to several days.
4. Complete dehydration in absolute alcohol for 24 hours.
5. Equal parts of pure ether and absolute alcohol, 24 hours.
6. Thin celloidin, 1-3 days.
7. Thick celloidin, 1-3 days.
8. Block. Harden block in 80 per cent alcohol.
9. Cut; keep sections in 80 per cent alcohol.
10. Stain; differentiate.
11. Wash thoroughly.
12. Dehydrate in 80 and 95 per cent alcohols.
13. Final dehydration and clearing in carbol-xylol.
14. Place on slide and blot with absorbent paper.
15. Mount in xylol-balsam or xylol-colophonium.

VIII. SUMMARY OF METHODS OF PREPARATION OF PARAFFIN SECTIONS.

1. Fixation of the tissue in alcohol, formol, etc.
2. Wash 24 hours, when necessary.
3. After-harden in 80 and 95 per cent alcohol for 1 to several days.
4. Complete the dehydration in absolute alcohol for 24 hours.
5. Aniline-oil until tissue becomes transparent.
6. 1st. Xylol, ½ hour, to remove aniline oil.
7. 2nd. Xylol, 1-2 hours, until translucent.
8. 1st. Paraffin (52°C.), ½ hour in oven, to remove xylol.
9. 2nd. Paraffin (52°C.), 1-12 hours in oven, until infiltrated.
10. Imbed and block; cool quickly.
11. Cut sections; mount on slides or covers.
12. Remove paraffin in xylol.
13. Remove xylol in absolute alcohol.
14. 80 per cent alcohol, for a few minutes.
15. Stain; differentiate; wash.
16. Dehydrate in 80 and 95 per cent alcohols.
17. Clear in carbol-xylol.
18. Mount in Canada-balsam or colophonium.

IX. ARTEFACTS IN MOUNTED SECTIONS.

A mounted section, after passing through the various stages indicated above, must of necessity present some appearances that are the result of the technical methods employed. The number and degree of such artefacts will depend upon the character of the methods employed and the care exercised in their performance. The trained observer ignores the presence of artefacts as having nothing at all to do with the significance of the section itself; but to the beginner in microscopic work they often appear to be the most important thing in the preparation, and are given a pathologic interpretation. How frequently do we see students, undergraduates and postgraduates, take up a section and pick out a fold, wrinkle, tear, staining-defect, precipitate, dirt, etc., as pathologic features! It is necessary, therefore, for the student to acquaint himself with the nature of artefacts so that he may ignore them and not give them an incorrect interpretation. The most important artefacts are as follows:—

1. =Artefacts due to fixation= (mercuric, chromic, osmic, etc.,
precipitates; alterations in blood-pigment due to formol; loosening
of cells from basement membrane due to contraction [kidney tubules,
etc.: loosened endothelium in blood-vessels, particularly confusing
to students]; destruction of red blood cells, as in alcohol
fixation; poor staining due to over-fixation).

2. =Artefacts due to hardening= (contraction, desquamation of
cells, etc.).

3. =Artefacts due to imbedding and cutting= (tears, holes,
ragged edges, irregular thickness, knife-streaks, compression of
soft structures, dislocation and tearing-out of firm tissues or
material, wrinkles, folds, etc.).

4. =Artefacts due to poor staining= (uneven, spotted or streaked
staining, overstaining, understaining, poor differentiation,
precipitates, insufficient washing, fading, poor contrasts,
defective staining due to presence of paraffin, dextrin, etc., in
section).

5. =Artefacts due to poor mounting= (imperfect dehydration and
clearing, cloudiness, milkiness or opacity of section; folds;
wrinkles; turned-over edges; tears in section caused by striking it
with balsam-dropper or needle; air-bubbles; lack of balsam).

6. =Dirt and foreign-material= (opaque and translucent dirt, above
or below section; coloring-matter in balsam; ink; pigment from
pencil; cotton-, silk, wool-, linen-, vegetable and paper-fibres,
hairs, desquamated squamous epithelium, portions of insects, etc.)