Adhesives and Mounting Media, Labeling 4.

Poly-L-Lysin e
• This aqueous detergent can be purchased as a 0.1%
Adhesives solution which is further diluted 1:10 with distilled
water (final dilution to 0.01%) prior to use.
• substances which can be smeared on to the slides so
• This is widely used as a section adhesive in
that the sections stick well to the slides.
immunohistochemistry.
• choice of slide and adhesive will be influenced by the
staining methods to be subsequently applied.
5. APES (3-aminopropylthriethoxysilane)
• not necessary for routine staining, provided that the
• APES-coated slides are very useful in cytology,
slides are clean and free from grease.
particularly for cytospin preparations of proteinaceous
• essential for methods that require exposure of sections
or bloody material.
to acids and alkalis (especially ammoniacal silver
• APES-coated slides are better than poly-L-lysine
solutions) during staining.
coated slides because they can be stored for a long
• This can be done either by leaving the slides in a 37°C
time without losing their adhesiveness.
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, Mounting
a longer drying time at lower temperature (e.g. 37°C for
• Last step in tissue processing that results in a
24 hours or longer) is recommended to avoid splitting
permanent histological preparation suitable for
and cracking of the section due to excess heat.
microscopy, after adhesion of the sections on to the
slide and appropriate staining of the tissue.

Instances when sections may float from the slide and

adhesives are necessary: Mounting Medium

• Urgent cryostat sections to be submitted for • Usually, a syrupy fluid applied between the section and
immunocytochemistry the coverslip after staining, setting the section firmly,
• Central nervous system tissues preventing the movement of the coverslip
• Tissues containing blood clot • Protects the stained section from getting scratched, to
facilitate easy handling and storage of the slides, and
• Tissues which have been decalcified
to prevent bleaching or deterioration due to oxidation,
• When sections are to be subjected to high
thereby preserving the slides for permanent keeping.
temperatures
• Often chosen for a specific refractive index (R.I.), which
1. Mayer's Egg Albumin can enhance specimen details or make them invisible.
• FORMULA:
o Egg White 50 cc. Refractive Index
o Glycerin 50 cc.
• Most commonly used because it is very easy to make, • Important because it governs the contrast between the
is convenient, and is relatively inexpensive. cellular detail and the background, and also the
transparency of the observed sample against the bright
2. Gelatin (1%) field of the microscope.
• FORMULA: • The mounting media must always have an RI higher
o Gelatin 1 gm than the mounted sample to impart more transparency.
o Distilled water 100 ml
o Glycerol 15 ml
Characteristics of a good mounting medium:
o Phenol Crystal 2 gm
• Adding up to 30 ml of 1% aqueous gelatin to the water • It should be colorless and transparent.
in a floating out bath and mixing it well is a most • It should be freely miscible with xylene and toluene.
convenient alternative to direct coating of slides. • It should not dry to a non-stick consistency and harden
relatively quickly.
3. Gelatin-formaldehyde mixture
• It should protect the section from physical damage and
• FORMULA: chemical activity (oxidation and changes in pH).
o 1% gelatin 5 ml
• It should be resistant to contamination (particularly
o 2% formaldehyde 5 ml
microorganism growth).
• Coat the slides with the above mixture. Allow coated
• It should not cause shrinkage and distortion of tissues.
slides to dry at 37°C for one hour or overnight before
• It should not leach out any stain or affect staining.
use.
• It should not change in color or pH.
• It should be compatible with the adhesive in use.
 • It should set without crystallizing, cracking or shrinking • Phosphate buffered glycerol
(or otherwise deform the tissue being mounted) and not (RI = 1.47)
react with, leach or induce fading in stains and reaction o Commonly used to mount sections for
products (including those from enzyme histochemical, immunofluorescence and glycerol may be added
hybridization, and immunohistochemical procedures). to other agents to retard drying and cracking.

• Glycerin jelly (Kaiser’s 1880) (RI: 1.47)

Labeling o FORMULA:
▪ Gelatin 10 gm.
• Slides should be properly labeled with an identifying ▪ Glycerol 70 ml.
case number on the side of the mounted coverslip to. ▪ Distilled water 60 ml.
• As a general rule, a paper label bearing the patient's ▪ Phenol crystals (preservative) 0.25 gm.
name, section number and preferably the staining o Glycerin jelly is the standard mounting medium
method used, is attached to the slide for proper used when dehydration and clearing with xylene
identification, while also avoiding any damage to the cannot be made (as in fat stains).
sections caused by wiping the "wrong" side of the slide.
• Farrant’s Medium (RI: 1.43)
o FORMULA:
Mounting media may be divided into two main groups: ▪ Gum arabic 50 gm.
▪ Distilled water 50 ml.
1. Aqueous Media ▪ Glycerol 50 ml.
• Aqueous mounting medium are used for mounting ▪ Sodium merthiolate 0.025 gm.
sections from distilled water when the stains would be o does not solidify upon storage and therefore
decolorized or removed by alcohol and xylene o does not need to be heated before use.
• made up of gelatin, glycerin jelly or gum arabic (to o takes a longer time to harden and may therefore
solidify the medium), glycerol (to prevent cracking require ringing.
and drying of the preparation), sugar (to increase the o Arsenic trioxide may be used as a substitute of
refractive-index), and a preservative solution. sodium merthiolate for preservation of the
medium.
• Water
o has a low refractive index, is moderately • Apathy’s medium
transparent and evaporates easily, hence is good o FORMULA:
only for temporary mounting. ▪ Pure gum arabic (crystals not powder) 50
o The refractive index of the water improves the gm.
image quality and also supports the specimen. ▪ Pure cane sugar or sucrose 50 gm.
o wet mounts cannot be stored over extended time ▪ Distilled water 50 ml.
periods, as the water evaporates. ▪ Thymol crystals 0.05 gm.
o used for methylene blue-stained nerve
• Glycerin (RI: 1.46) preparations and as a general purpose aqueous
o may also be used as a preservative. mountant.
o has a high index of refraction and provides o one of the most useful aqueous mountants for
greater visibility if slightly diluted with water (for fluorescent microscopy, being virtually non-
moist sections). fluorescent.
o very suitable semi-permanent mounting medium o sets quite hard, has a higher refractive index, and
o miscible with water, is inexpensive, and is non- does not require ringing.
poisonous.
o Disadvantages of Glycerin • Brun’s Fluid
▪ difficult to prepare slides that are truly o FORMULA:
permanent in nature. ▪ Glucose 24 gm.
▪ slowly run off a slide that is tilted. ▪ Glycerine 6 ml.
▪ Do not stack slides for long as the ▪ Spirits of camphor 6 ml.
pressure will squeeze glycerin from the ▪ Distilled water 84 ml.
mounts. o Brun's Fluid is recommended for mounting frozen
▪ Glycerin will eventually evaporate and air sections from water.
will penetrate under the coverslip.
▪ Glycerin can be attacked by 2. Resinous mounting media
microorganisms. • 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 (RI = 1.524)
o Canadian tree: Abus Balsamea
o dissolved in xylene in an incubator at 37°C or
paraffin oven at 58 °C
o It is a transparent, almost colorless oleoresin that
adheres firmly to glass and sets to a hard
consistency without granulation.
o Calcium carbonate chips may be added to
maintain its neutral reaction.
o Canada balsam is recommended for whole
mounts and for thick sections because it does not
shrink much.

• DPX - (Dibutyl Phthalate and Xylene) (RI = 1.532)

o recommended for small tissue sections but not
for whole mounts because of shrinkage produced
on drying
o colorless, neutral medium in which most standard
stains are well preserved.
o It tends to set quickly and, in doing so, often
retract from the edge of the coverslip.

• XAM (RI = 1.52)

o Xam is a synthetic resin mixture in xylene,
available in a pale yellow or colorless solution.
o Dries quickly without retraction, and preserves
stains well.

• CLARITE (RI = 1.544)

o synthetic resin which is soluble in xylene
o generally preferred over D.P.X

Other Recommended Synthetic Mounting Media

• Permount (made by Fisher Scientific)

• H.S.R. (Harleco Synthetic Resin)
• Clearmount (Gurr)

Ringing

• 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.”
• Kronig cement - made up of two parts paraffin wax
mixed with 4-9 parts powdered colophonium resin,
heated and filtered.
• Durofix - cellulose adhesives