LEC#12 HISTOPATHOLOGY
PROF. Dr. Marissa A. Cauilan
LEC
ADHESIVES AND MOUNTING MEDIA
OUTLINE
I. Adhesives
a. Mayer’s Egg Albumin
b. Dried Albumin
c. Gelatin (1%)
d. Gelatin-Formaldehyde Mixture
e. Poly-L-Lysine
f. APES
II. Mounting Medium
Characteristics of a Good Mounting
A. Aqueous Mounting Medium
A.1. Water
A.2. Glycerin
A.3. Farrant’s Medium
A.4. Apathy’s Medium
A.5. Brun’s Fluid
B. Resinous Mounting Media
B.1. Canada Balsam
B.2. DPX
B.3. XAM
B.4. Clarite
III. Mountants for Immunochemical Staining
IV. Cover Slipping
V. Ringing
VI. Broken Slides
• Mounting is the last step in tissue processing which
leads in a permanent histological preparation suitable for
microscopy, after adhesion of the sections on to the slide
and appropriate staining of the tissue
• Sections are floated our on a water-bath after cutting.
Bubbles accumulating under the ribbon may be removed
using a smooth teasing needle care being taken in order
not to tear the section. Bubbles may also be removed
through 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 already 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 37oC
incubator overnight, by placing the slides in a wax oven at
56o to 60oC for 2 hours, or by drying the slides on a hot
plate at 45o to 55oC for 30 to 45 minutes
• For more delicate tissues, such as the CNS tissue or
brain, a longer drying time at lower temperature (e.g.,
37oC for 24 hours or longer) is recommended to avoid
splitting and cracking of the section caused by excess
heat
• Another alternative to drying is through 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
I. ADHESIVES
• It is a substance which can be smeared on to the slides in
order for the sections to stick well to slides
• The choice of slide and adhesive to be used will be
influenced by the staining methods to be subsequently
applied
• Slides must always be grease-free and dust-free and
stores and handled correctly
• Slides must always be accurately and appropriately
labelled in a manner compliant with provided local
regulations
• If staining is to include antigen retrieval (IHC), enzyme
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pretreatment for in-situ hybridization (ISH), or prolonged
incubation steps, charged slides or an adhesive must be used.
Some special stains, particularly those than employ alkaline
reagents, can also cause sections to lift
• Adhesives are not necessary to be utilized in routine
staining, provided that the slides are clean and free from
grease
• Adhesives 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 adhesive used and applied on the slide should be kept
to a minimum, because they ae 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:
o For urgent cryostat sections to be submitted for
immunocytochemistry
o For central nervous system tissues
o For tissues containing blood clot
o For tissues which have been decalcified
o When sections are to be subjected to high
temperatures
• Albumin is the most commonly used adhesive
• 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
• Thymol resistant organisms growing in the adhesive
have been known to contaminate gram-stained sections
and cause confusion during microscopic examination
• Disadvantage of Albumin:
- it retains some of the stain and gives a dirty
background
• Poly-L-lysine, which is 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
diluted with 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
period of 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
MAYER’S EGG ALBUMIN
• It is the most commonly used because it is very easy to
make, is convenient, and is relatively inexpensive
• Formula:
o Egg White 50 cc.
o Glycerin 50 cc.
o Filter and add about 100 mg crystals of thymol to
prevent the growth of molds
• A drop of Mayer’s egg albumin is usually smeared into a
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
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ADHESIVES AND MOUNTING MEDIA
• 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
DRIED ALBUMIN
• It is dried and stored in 70% alcohol until it is ready
for staining
• Formula:
o Dried albumin 5 gm
o Sodium chloride 5 gm
o Dissolve in 100 cc. of distilled water and add
crystals of thymol
GELATIN (1%)
• Formula:
o Gelatin 1 gm
o Distilled water 100 ml
o Glycerol 15 ml
o 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
GELATIN-FORMALDEHYDE MIXTURE
• Formula:
o 1% Gelatin 5 ml
o 2% Formaldehyde 5 ml
• Coat the slides with the above mixture. Allow the coated
slides to dry at 37oC for one hour or overnight before use
POLY-L-LYSINE
• It is an aqueous detergent that 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 distilled poly-L-lysine and
allowed to dry
• It is widely used as a section adhesive in
immunocytochemistry
• Poly-L-Lysine coated slides must be used within a few
days after they are prepared because its effectiveness as
an adhesive slowly decreases in time
APES (3-AMINOPROPYLTHIETHOXYSILANE)
• 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
• The slides 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
II. MOUNTING MEDIUM
 Bond specimen, slide and coverslip together with a clear
durable film
 Usually, a syrupy fluid applied between the section and
the coverslip after staining, setting the section firmly,
preventing the movement of the coverslip
 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
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 Helps prevent the distortion of image during microscopic
examination
 Often chosen for a specific refractive index (R.I.), which
can enhance specimen details or make them invisible
o 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.
Note:
 Do not use immersion oil on an uncovered slide.
 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.
 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.
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).
AQUEOUS MOUNTING MEDIA
• It is 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
• It is 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
1. WATER
• Low refractile index
• Moderately transparent
• Evaporates easily
• Good only for temporary mounting
• Does not allow tissues to be examined under the oil
immersion lens
NOTE:
In a wet mount, the specimen is suspended in a drop of
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ADHESIVES AND MOUNTING MEDIA
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
 It may also be used as a preservative
 Has a high index of refraction
 Provides greater visibility if slightly diluted with water (for
moist sections)
 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
 Miscible with water, inexpensive, non-poisonous
NOTE:
Glycerin can be attacked by microorganisms, so one can
optionally add a crystal of thymol to avoid bacteria and
fungi.
GLYCERIN JELLY (KAISER'S 1880)
• Refractive Index 1.47
• FORMULA:
o Gelatin 10 gm.
o Glycerol 70 ml.
o Distilled water 60 ml.
o Phenol crystals (preservative) 0.25 gm.
NOTE:
• 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
. 
3. FARRANT’S MEDIUM
 Refractive Index: 1.43
• FORMULA:
o Gum arabic 50 gm.
o Distilled water 50 ml.
o Glycerol 50 ml.
o Sodium Merthiolate 0.025 gm.
• Dissolve gum arabic in distilled water with gentle heating
and add glycerol and sodium merthiolate. Mix well and
label.
• The 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:
o Pure gum arabic (crystal not powder) 50 m
o Pure cane sugar or sucrose 50 m
o Distilled water 50 ml
o Thymol crystal 0.05
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 This medium is used for methylene blue-stained
nerve preparations and as a general-purpose aqueous
mountain
 It is one of the most useful aqueous mountants
for fluorescent microscopy, being virtually non-
fluorescent
 Dissolve the ingredients with the aid of gentle heat
 Von apathy’s medium is not compatible with normal
histologic stains
 The pH of the medium is near 4.0 (highly acidic), so
stains fade or bleed into medium
NOTE:
Addition of 50 grams potassium acetate for 10 grams
sodium chloride will prevent bleeding of metachromatic
stains for amyloid.
5. BRUN’S FLUID
 It 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
this agent
• FORMULA:
o Glucose 24 gm
o Glycerin 6 ml
o Spirits of camphor 6 ml
o Distilled water 84 ml
RESINOUS MOUNTING MEDIA
• It is used for preparations that have been dehydrated
and cleared in xylene or toluene and are recommended
for majority of staining methods
• It can be classified as natural or synthetic resins
• The most important synthetic resins are used for
embedding undecalcified bones, and for electron
microscopy
1. CANADA BALSAM
Refractive Index: 1.524
 It is a natural resin extracted from the Canadian tree,
Abus Balsamea, usually dissolved in xylene in an
incubator at 37oC or paraffin oven at 58oC, and filtered,
obtaining the desired consistency by controlled
evaporation of the solvent
 The solution can be made neutral or acidic 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 transparent, almost colorless oleoresin that adheres
firmly to glass and sets to a hard consistency without
granulations
 It darkens slightly with age and slowly becomes acid
because it oxidized xylene, thereby causing gradual
fading of many stains
 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 may be less of a health hazard but may
cause other problems such as slow gardening 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
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ADHESIVES AND MOUNTING MEDIA
 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 fur 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; however, it is quite
expensive
 It 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, clearing agents mixed
with xylene, pure xylene, and balsam dissolved in xylene
 Toluene or benzene could be used instead of xylene.
2. DIBUTYL PHTHALATE AND XYLENE
 Refractive Index: 1.532
 It 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 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
3. XAM
 Refractive Index: 1.52
 It is a synthetic resign 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
4. CLARITE (CLARITE X)
 Refractive Index: 1.544
 It is a synthetic resin which is soluble in xylene (it is used
as a 60% solution in xylene), and is generally preferred
over DPX
 Other recommended synthetic media include: (1)
Permount, made by Fisher Scientific, (2) H.S.R. or
Harleco Synthetic Resin, and (3) Clearmount (Gurr)
III. MOUNTANTS FOR IMMUNICHEMICAL STAINING
 The choice of mounting medium following
immunochemical staining is largely dictated by the label,
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 aqueous mounting 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
 Exposure to excitation light of most fluorescent labels
results in diminished staining, a process known as photo
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bleaching
 Apathy’s medium (RI of 1.52) is the most useful aqueous
mountant for fluorescent microscopy, being virtually non-
fluorescent
 Polyvinyl alcohol (RI of 1.5) is an alternative for glycerin
jelly, commonly used for fluorescent labels with
paraphenylene-diamine as antifading agent
 Ready-to-use anti-fading kits are also commercially
available
IV. COVER SLIPPING
 The stained section on the slide must be covered with a
thin piece of plastic or glass in order 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
 Bubbled 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 of cover slipping media
may also produce a bubbled appearance under the
microscope
V. RINGING
 It 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 like Durofix
VI. BROKEN SLIDES
 Mounting a broken slide on to another clean xylene-
moist slide with a drop of mounting media (Clarite or
Premount) may be sufficient for immediate examination
while a new section is being cut and stained
 If an important 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 37oC 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 37oC incubator until dry, washed
gently with butyl acetate, then washed well with xylene,
and mounted in Clarite or Permoun.
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