CHAPTER

15
ADHESIVES AND MOUNTING MEDIA
Mounting is the last step in tissue processing that results in a permanent
histological preparation suitable for microscopy, after adhesion of the sections
on to the slide and appropriate staining of the tissue.
After cutting, sections are floated out on a water-bath. Bubbles
accumulating under the ribbon may be removed with a smooth teasing needle,
care being taken not to tear the section. Bubbles may also be removed by
pulling the ribbon very gently across the long edge of a glass slide held below
the section in the water bath. When the sections chosen have flattened out, the
numbered slide is immersed in the water bath and the section is fished out and
drained.
After draining, the sections are fixed to the slides. This can be done either
by leaving the slides in a 37°C incubator overnight, by placing the slides in a
wax oven at 56° to 60°C for 2 hours, or by drying the slides on a hot plate at
45° to 55°C for 30 to 45 minutes. For more delicate tissues like the CNS tissue
or brain, a longer drying time at lower temperature (e.g. 37°C for 24 hours or
longer) is recommended to avoid splitting and cracking of the section due to
excess heat.
Another alternative to drying is by the use of adhesives. To promote
adhesion of sections, adhesives may be spread thinly and evenly on a clean
grease-free slide which is then gently approximated to the end of the ribbon,
and drawn upwards in a near vertical motion.

ADHESIVES
An adhesive is a substance which can be smeared on to the slides so that
the sections stick well to the slides. The choice of slide and adhesive will be
influenced by the staining methods to be subsequently applied. Slides must
always be grease- and dust- free and stored and handled correctly. If staining is
to include antigen retrieval (IHC), enzyme pretreatment for in-situ
hybridization (ISH), or prolonged incubation steps, charged slides or an
adhesive must be used. Some special stains, particularly those that employ
alkaline reagents, can also cause sections to lift. Slides must always be
accurately and appropriately labelled in a manner compliant with local
regulations.
Adhesives are not necessary for routine staining, provided that the slides
are clean and free from grease. However, they are essential for methods that
require exposure of sections to acids and alkalis (especially ammoniacal silver
solutions) during staining. In such cases, the amount of adhesives applied on
the slide should be kept to a minimum, since they are prone to contamination
and bacterial growth that might be confused with real tissue organisms
demonstrated by Gram and PAS stains.
If clean grease-free slides are used and sections are adequately dried, the
sections will not float off during staining and adhesive will not be necessary.
There are still certain instances when sections may float from the slide:
For urgent cryostat sections to be submitted for
immunocytochemistry
For central nervous system tissues
For tissues containing blood clot
For tissues which have been decalcified
When sections are to be subjected to high temperatures
The most commonly use adhesive is Albumin. Albumin solution is
prepared by mixing equal parts of glycerin, distilled water and white of eggs,
then filtered through coarse filter paper and a crystal of Thymol is added. One
disadvantage of using albumin is that it retains some of the stain and gives a
dirty background. Thymol resistant organisms growing in the adhesive have
been known to contaminate gram-stained sections and cause confusion during
microscopic examination.
Poly-L-lysine, also a favorite adhesive, can be bought as a 0.1 % solution
and further diluted (1 in 10 with distilled water) when ready to use. Sections are
coated with this dilute poly-L-lysine and allowed to dry. With time, the
adhesive ability of this substance slowly loses its effectiveness. Therefore the
coated slides should be used within a few days.
Aminopropyltriethoxysilane (APES) is a better section adhesive and
coated slides can be stored for a long time. Slides are dipped in 2% APES in
acetone drained then dipped in acetone, drained again and finally dipped in
distilled water. It is invaluable in cytology particularly for cytosine preparation
of proteinaceous or bloody material.

1. Mayer's Egg Albumin
FORMULA:
Egg White 50 cc.
Glycerin 50 cc.
Filter and add about 100 mg. crystals of thymol to prevent the
growth of molds.

 Mayer's egg albumin is the most commonly used because it is very easy to
make, is convenient, and is relatively inexpensive. A drop of Mayer's egg
albumin is usually smeared into the clean glass slide before sections are
oriented. Sections which have been creased on cutting may be stretched by
gentle heating before attaching them into slides. During staining, the excess of
albumin may also take up the stain and interfere with diagnosis; hence, it
should be wiped off from the slide to remove any excessive solution.
For celloidin sections, egg albumin is smeared on the slide. The section is
then transferred from 95% alcohol bath to the slide, pressed flat on the slide with
a smooth filter paper coated with thin celloidin mixture.

2. Dried Albumin - dried, and stored in 70% alcohol until it is ready for
staining.
FORMULA
Dried albumin 5 gm
Sodium chloride 5 gm
Dissolve in 100 cc. of Distilled Water and add crystals of thymol.

3. Gelatin (1%)
FORMULA:
Gelatin 1 gm
Distilled water 100 ml
Glycerol 15 ml
Phenol Crystal 2 gm
Adding up to 30 ml of 1% aqueous gelatin to the water in a floating
out bath and mixing it well is a most convenient alternative to
direct coating of slides.

4. Gelatin-formaldehyde mixture
FORMULA:
1% gelatin 5 ml
2% formaldehyde 5 ml
Coat the slides with the above mixture. Allow coated slides to dry at
37°C for one hour or overnight before use.

5. Poly-L-Lysin e
This aqueous detergent can be purchased as a 0.1% solution which is
further diluted 1:10 with distilled water (final dilution to 0.01%) prior to
 use. Sections are coated with this diluted poly-L-lysine and allowed to
dry. This is widely used as a section adhesive in immunohistochemistry.
Poly​L-lysine coated slides must be used within a few days after they are
prepared, since its effectiveness as an adhesive slowly decreases in time.

6. APES (3-aminopropylthriethoxysilane)
APES-coated slides are very useful in cytology, particularly for
cytospin preparations of proteinaceous or bloody material. The slides are
dipped in 2% APES in acetone, drained, dipped in acetone, drained again,
and finally dipped in distilled water. They are then placed upright in a
rack and allowed to dry. APES-coated slides are better than poly-L-lysine
coated slides because they can be stored for a long time without losing
their adhesiveness.


MOUNTING MEDIUM

If an unmounted stained section is seen in the microscope, differences in
light refraction between the glass slides and the tissue components, may lead to
difference in length dispersion; hence, very little microscopic detail can usually
be appreciated. Tissues should therefore be impregnated with a transparent
medium that has an index of refraction close to that of the glass and the tissue.
The mounting medium bonds specimen, slide and coverslip together with a
clear durable film.
A mounting medium is usually a syrupy fluid applied between the section
and the coverslip after staining, setting the section firmly, preventing the
movement of the coverslip. It protects the stained section from getting
scratched, to facilitate easy handling and storage of the slides, and to prevent
bleaching or deterioration due to oxidation, thereby preserving the slides for
permanent keeping. The mounting medium also helps prevent the distortion of
image during microscopic examination.
Mounting media are often chosen for a specific refractive index (R.I.),
which can enhance specimen details or make them invisible. Materials like
glass become totally invisible if immersed in a solution of the same refractive
index. Refractive index is important because it governs the contrast between
the cellular detail and the background, and also the transparency of the
observed sample against the bright field of the microscope. The mounting
media must always have an RI higher than the mounted sample to impart more
transparency.
The slide carrying the section to be mounted is taken from the last xylene
bath with the forceps. Excess xylene is wiped off from the back of the slide and
from around the section. A drop of mounting medium is placed down the center
of the slide. A clean, dry cover glass is placed on the edge of the slide and
gradually inclined downward until it touches the mounting medium and gently
pressed on to the slide while the mounting medium quickly spreads through the
whole area of the section. The slide may then be incubated at 37°C for 12-24
hours after mounting, to harden the medium.
Do not use immersion oil on an uncovered slide. If in a hurry to view a
specimen under oil immersion, mount the slip and handle the mount carefully,
keeping it horizontal. Oil can be applied, but do not attempt to wipe it off until
the mounting medium is cured - at least overnight or an hour on a hotplate or in
an oven. Do not store mounted slides vertically for 2 days if cured at room
temperature.
Excessive mounting medium will cause it to ooze out of the sides of the
cover glass, and should be carefully wiped with a fine cloth moistened with
xylene. Excessive blotting, on the other hand, will dry up the section, causing
shrinkage and cracking of the specimen. If the section has to be remounted, the
cover glass may be removed by soaking in xylene. Excess xylene, if not
removed, will mix with the mountant and form bubbles on the slide. Too little
mounting medium may also cause improper setting of the coverslip or
formation of bubbles on the section, which can be teased out by gently pressing
on the cover glass with a pointed forceps or mounting needle. Setting may be
hastened in a hot oven at 50°C for 2 hours.

Characteristics of a good mounting medium:
1. It should be colorless and transparent.
2. It should be freely miscible with xylene and toluene.
3. It should not dry to a non-stick consistency and harden relatively
quickly.
4. It should protect the section from physical damage and chemical
activity (oxidation and changes in pH).
5. It should be resistant to contamination (particularly microorganism
growth).
6. It should not cause shrinkage and distortion of tissues.
7. It should not leach out any stain or affect staining.
8. It should not change in color or pH.
9. It should be compatible with the adhesive in use.
 10. It should set without crystallizing, cracking or shrinking (or
otherwise deform the tissue being mounted) and not react with, leach
or induce fading in stains and reaction products (including those
from enzyme histochemical, hybridization, and
immunohistochemical procedures).

A mounting medium should be chosen that will not fade the particular
stains used; for example, basic aniline dyes should be mounted in non-acid
containing mountants. Preparations showing the Prussian blue reaction should
be mounted in non-reducing media. The mounting medium is usually
dispensed from the stock bottles into screw cap collapsible tin tubes. This will
keep the mountant clean and prevent the concentration and thickening of the
solution subsequent to evaporation.
Slides should be properly labeled with an identifying case number on the
side of the mounted coverslip to. As a general rule, a paper label bearing the
patient's name, section number and preferably the staining method used, is
attached to the slide for proper identification, while also avoiding any damage
to the sections caused by wiping the "wrong" side of the slide.

Mounting media may be divided into two main groups:
a. Aqueous Media
b. Resinous Media

AQUEOUS MOUNTING MEDIA
Aqueous mounting medium are used for mounting sections from distilled
water when the stains would be decolorized or removed by alcohol and xylene
as would be the case with most of the fat stains (Sudan methods) or for
metachromatic staining of amyloid. They are usually made up of gelatin,
glycerin jelly or gum arabic (to solidify the medium), glycerol (to prevent
cracking and drying of the preparation), sugar (to increase the refractive-index),
and a preservative solution.

Following are examples of common aqueous mounting media:
1. Water - has a low refractive index, is moderately transparent and
evaporates easily, hence is good only for temporary mounting. Also, water
does not allow tissues to be examined under the oil immersion lens. In a
wet mount, the specimen is suspended in a drop of liquid (usually water)
 located between slide and cover glass. The water refractive index of the
water improves the image quality and also supports the specimen. In
contrast to permanently mounted slides, wet mounts cannot be stored over
extended time periods, as the water evaporates.
2. Glycerin - may also be used as a preservative. It has a high index of
refraction and provides greater visibility if slightly diluted with water (for
moist sections). This is a very suitable semi-permanent mounting medium
with a refractive index of 1.46, sets quite hard, and will keep sections
mounted for years, especially if sealed on the edges with paraffin wax. It is
miscible with water, is inexpensive, and is non-poisonous. It is also not
necessary to treat the specimens with alcohol or organic solvents, which
may introduce artifacts and remove pigments. This is usually regarded as
the standard mountant for fat stains.
The disadvantage is, that it is difficult to prepare slides that are truly
permanent in nature. Because it is a thick liquid, it can slowly run off a
slide that is tilted. A proper sealing of the cover slip corners is absolutely
necessary if one wants to store the slides over extended periods. Do not
stack slides for long as the pressure will squeeze glycerin from the mounts.
Glycerin will eventually evaporate and air will penetrate under the cover
slip. Glycerin can be attacked by microorganisms, so one can optionally
add a crystal of thymol to avoid bacteria and fungi.
As with other solvents it is used because it is cheap, safe and quick to
use with little preparation. Ringing the coverslip with a hydrophobic seal
will extend the life of mounted sections, although cationic dyes will diffuse
into the medium over time. Phosphate buffered glycerol (RI = 1.47) is
commonly used to mount sections for immunofluorescence and glycerol
may be added to other agents to retard drying and cracking.

GLYCERIN JELLY (KAISER'S 1880)
(Refractive Index 1.47)
FORMULA:
Gelatin 10 gm.
Glycerol 70 ml.
Distilled water 60 ml.
Phenol crystals (preservative) 0.25 gm.
Gelatin is added to distilled water and incubated in a water bath at
 60°C until dissolved. Glycerol and then phenol crystals are added and
mixed. The solution is labeled, and stored in a refrigerator at 40°C. For
use, the gelatin must be heated in a water bath or incubator at 60°C to
melt. Too much gelatin makes the jelly difficult to melt and included
bubbles found on the slide will not burst. The melted medium should
not be shaken or stirred before use, if formation of air bubbles is to be
avoided.
Glycerin jelly is the standard mounting medium used when dehydration
and clearing with xylene cannot be made (as in fat stains). Pure glycerin has the
highest index of refraction and thus provides the best viewing and may be
optimal for critical or irreplaceable material, because old material, when
glycerin is mostly evaporated, is easily retrieved with hot water or steam. The
disadvantage is that it should be melted before use (due to the presence of
gelatin). Stains mounted on glycerin jelly tend to fade. Polyvinyl alcohol, often
used as a mountant in immunofluorescence microscopy, has been
recommended as an alternative for glycerine jelly. The mountant is not set in
the desired amount of hardness and therefore requires "ringing".

3. FARRANT'S MEDIUM
(Refractive Index 1.43)
FORMULA:
Gum arabic 50 gm.
Distilled water 50 ml.
Glycerol 50 ml.
Sodium merthiolate 0.025 gm.
Dissolve gum arabic in distilled water with gentle heating and add
glycerol and sodium merthiolate. Mix well and label.
This gum arabic medium does not solidify upon storage and therefore
does not need to be heated before use. However, it takes a longer time to
harden and may therefore require ringing.
Arsenic trioxide may be used as a substitute of sodium merthiolate for
preservation of the medium. Addition of 50 gm. potassium acetate will
produce a neutral (pH 7.2) instead of an acid (pH 4.4) medium, and
therefore, will raise the refractive index to 1.44.

4. APATHY'S MEDIUM
(Refractive Index 1.52)
FORMULA:
 Pure gum arabic (crystals not powder) 50 gm.
Pure cane sugar or sucrose 50 gm.
Distilled water 50 ml.
Thymol crystals 0.05 gm.

This medium is used for methylene blue-stained nerve preparations
and as a general purpose aqueous mountant. It is one of the most useful
aqueous mountants for fluorescent microscopy, being virtually non-
fluorescent. Von Apathy’s medium is not compatible with normal
histological stains. The pH of the medium is near 4.0 (highly acidic) so
stains fade or bleed into the medium. Addition of 50 grams potassium
acetate, 20 grams of calcium chloride or 10 grams sodium chloride can
raise the pH to near 7.0 and will prevent "bleeding" of metachromatic
stains for amyloid. The medium sets quite hard, has a higher refractive
index, and does not require ringing.

5. BRUN'S FLUID
FORMULA:
Glucose 24 gm.
Glycerine 6 ml.
Spirits of camphor 6 ml.
Distilled water 84 ml.
Mix, shake well, and filter. Store the solution in a well-stoppered
bottle.
Brun's Fluid is recommended for mounting frozen sections from
water.
Frozen sections that are mounted directly from water or paraffin
sections which require dehydration and clearing, usually should be
mounted on glycerin, gum syrup or Brun's fluid.


RESINOUS MOUNTING MEDIA
Sections of tissue embedded in plastic compounds (such as epoxy resins)
can be successfully mounted in liquid resin of the same type. Sections should
be completely dry before applying mountant, which is best set using the same
conditions prescribed for tissue blocks. Resinous media are used for
preparations that have been dehydrated and cleared in xylene or toluene, and
are recommended for majority of staining methods. They may be divided into
natural and synthetic resins. The most important synthetic resins are used for
embedding undecalcified bones, and for electron microscopy.

Canada Balsam
(Refractive Index 1.524)
Canada Balsam is a natural resin extracted from the Canadian tree, Abus
Balsamea, usually dissolved in xylene in an incubator at 37°C or paraffin oven
at 58 °C, and filtered, obtaining the desired consistency by controlled
evaporation of the solvent. The solution can be made neutral or acid by adding
excess amounts of calcium carbonate or salicylic acid, allowing the mixture to
settle, decanting the supernatant liquid into a stock bottle, and discarding the
residue.
It is a transparent, almost colorless oleoresin that adheres firmly to glass
and sets to a hard consistency without granulation. However, it darkens slightly
with age and slowly becomes acid because it oxidizes xylene, thereby causing
gradual fading of many stains.
The harmful solvents which, constitute a health hazard such as xylene,
may limit the use of Canada Balsam as a mounting medium. The use of non-
toxic solvents like histomount instead of xylene like histomount may be less of
a health hazard but may cause other problems such as slow hardening and
premature darkening. Benzene may be substituted for xylene as a solvent. The
medium can only be neutralized temporarily since the mixture becomes acidic
and changes into a brown color upon storage. Calcium carbonate chips may be
added to maintain its neutral reaction. The solution acidifies and darkens with
age and upon exposure to sunlight, for which reason it should be kept in a dark
glass bottle. Stains are usually not preserved due to acidity on prolonged
exposure.
Canada balsam is recommended for whole mounts and for thick sections
because it does not shrink much. It sets hard without granulation; it is,
however, quite expensive. As Canada Balsam does not mix with water,
mounting in it implies the use of a sequence of dehydration, starting with low
grade alcohols, followed by high grade alcohols, absolute alcohol, mixed
clearing agents plus alcohol, clearing agents, clearing agents mixed with
xylene, pure xylene, and balsam dissolved in xylene. Toluene or benzene could
be used instead of xylene.

DPX - (Dibutyl Phthalate and Xylene)
(Refractive Index 1.532)
This is a resinous medium recommended for small tissue sections but not
for whole mounts because of shrinkage produced on drying; hence, it should be
used in excess amounts. It is a colorless, neutral medium in which most
standard stains are well preserved. It is prepared by dissolving the common
plastic, polystyrene, in a suitable hydrocarbon solvent (usually xylene). It tends
to set quickly and, in doing so, often retract from the edge of the coverslip. It
has a greater advantage over Canada balsam in that slides can be cleaned of
excess mountant simply by stripping it off after cutting around the edge of
coverslip.

XAM
(Refractive Index 1.52)
Xam is a synthetic resin mixture in xylene, available in a pale yellow or
colorless solution. It dries quickly without retraction, and preserves stains well.
Sections are quickly mounted from xylene.


CLARITE
(Refractive Index 1.544)
Clarite (or Clarite X) is a synthetic resin which is soluble in xylene (it is
used as a 60% solution in xylene), and is generally preferred over D.P.X. Other
recommended synthetic mounting media include Permount (made by Fisher
Scientific), H.S.R. (Harleco Synthetic Resin), and Clearmount (Gurr).

Mountants for immunochemical staining
The choice of mounting medium following immunochemical staining is
largely dictated by the label (and in the case of enzymatic labels, the
chromogen) used to visualize the antigen. Aqueous mounting medium is
generally suitable for all enzymatic label/chromogen combinations and
fluorescent labels. Specimens mounted in such media are mounted straight
from the aqueous phase (with no dehydration or clearing).
Aqueous mounting media for phycobiliprotein fluorescent labels
(phycoerythrin, phycocyanin) must not contain glycerol as this quenches the
staining intensity. Similarly, exposure to excitation light of most fluorescent
labels results in diminished staining, a process known as photo bleaching.
Apathy’s medium (Refractive Index 1.52) is the most useful aqueous mountant
for fluorescent microscopy, being virtually non-fluorescent. Polyvinyl alcohol
(Refractive Index 1.5) is an alternative for glycerine jelly, commonly used for
fluorescent labels with paraphenylene-diamine as antifading agent. Ready-to-
use anti-fading kits are also commercially available.

Cover slipping
The stained section on the slide must be covered with a thin piece plastic or
glass to protect the tissue from being scratched, to provide better optical quality
for viewing under the microscope, and to preserve the tissue section for years to
come. The stained slide must go through the reverse process that it went through
from paraffin section to water. The stained slide is taken through a series of
alcohol solutions to remove the water, then through clearing agents to a point at
which a permanent resinous substance beneath the glass coverslip, or a plastic
film, can be placed over the section.
Bubbles under the coverslip may form when the mounting media is too thin, and
as it dries air is sucked in under the coverslip. Contamination of clearing agents
or cover slipping media may also produce a bubbled appearance under the
microscope.

Ringing
Ringing is the process of sealing the margins of the cover-slip to prevent
the escape of fluid or semi-fluid mounts and evaporation of mountant, to fix the
coverslip in place, and to prevent sticking of the slides upon storage. The term
“ringing” originated because round coverslips were initially used and the
coating applied in the form of a circle or “ring.” A liquid preparation sealed
well with nail polish could last some months. Paraffin wax may be applied with
a ringing iron and is satisfactory as a temporary ringing agent. The ringing
media used may be Kronig cement made up of two parts paraffin wax mixed
with 4-9 parts powdered colophonium resin, heated and filtered. Also available
are cellulose adhesives such as Durofix.

Broken Slides
Mounting a broken slide on to another clean xylene-moist slide with a drop
of mounting media (Clarite or Permount) may be sufficient for immediate
examination while a new section is being cut and stained. If an important slide
is broken and replacement is not available, the section (if still intact) may be
transferred to another slide.
The coverslip can be removed by soaking in xylene, and placing the
broken slide in the incubator at 37°C until all the mountant has been removed.
The whole slide is then covered with a mixture of 6 parts butyl acetate and 1
part durofix and left in the incubator for 30 minutes until the mixture hardens
into a film. Using a sharp scalpel blade, the hardened film is cut around the
section, and the slide is placed in cold water until the film and section float off.
The film containing the section is mounted on a clean slide, placed in the 37°C
incubator until dry, washed gently with butyl acetate, then washed well with
xylene, and mounted in Clarite or Permount.

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