Mounting Media

Dr Iram Iqbal

Aim
To define and discuss different type of mounting medias ,their technique for mounting sections ,and the qualities of the ideal mountant.

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Defination Qualities of an ideal mountant Classification of mounting media Cover glasses Properties of the good mounted sections Technique for mounting sections Labelling & and storage of slides

MOUNTING OF SECTIONS

After staining and before examination, sections should be mounted with a suitable medium under cover slips . To facilitate ease of handling and storage and to prevent damage to the section. The liquid used for mounting slides is known as “Mountant”.

Qualities of an ideal mountant

An ideal mounting medium must fulfill following criteria;  It should fulfill the basic requirement of protecting from dust and damage.  It should preserve the cell characteristics and quality of the stain.  It should be colourless and transparent .

AN IDEAL MOUNTANT

The medium should have a refractive index as close as possible to that of the glassi,e,; 1.5 . It should not cause stains to “bleed”or fade It should not crack or appear granular on setting. It should be non sticky and harden quickly.

Cont…

It should not shrink back from the edges of the cover glass. It must be free flowing. It must be free from bubbles. It should be resistant to contamination Once set, the mountant should remain stable.

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Types of mounting Media

Aqueous based mounting media Resinous mounting media .

AQUEOUS MOUNTANT

Apathy,s Medium (RI 1.52) Farrant,s Medium (RI 1.43) Fructose (Levulose) Syrup (RI 1.47) Glycerin Jelly (RI i.47) Highman,s Medium (RI 1.52)

RESINOUS MOUNTANTS
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Canada balsam (RI 1.52) Cover bond (RI I.53) DePeX (RI 1.52) Gurr,s Neutral Mounting Medium(RI 1.51 ) Histoclad( RI 1.54) Permount (RI 1.526) Pro-Texx (RI 1.495) Technicon Resin (RI 1.62) UVinert (RI 1.517) XAM (RI 1.52)

It is important to note also that the clearing agent used must be compatible with the mounting medium, or the sections must be thoroughly dried prior to mounting.

Aqueous Mounting Media
Aqueous mounting media are used for mounting sections from distilled water.

AQUEOUS MOUNTING MEDIA

It is used in cases where the stain would be decolourized or removed by alcohol and xylene Aqueous mountants require the addition of a bacteriostatic agent such as phenol to prevent the growth of molds Once the medium has hardened, a permanent seal can be obtained by ringing the coverglass with clear nail polish

Apathy’s Medium (RI 1.52) It is a very useful medium for mounting sections for fluorescent microscopy.  Gum Arabic 50g  Sucrose 50g  Distilled water 50ml  Thymol 1 crystal This medium remains fluid in storage and hardens by evaporation . Should be stored in well stoppered bottle.

Farrant`s Medium (RI1.43)
Gum Arabic Glycerin Distilled water Sodium merthiolate 50g 50ml 50ml 0.025g

This medium remains fluid in storage there for more convenient than glycerin jelly. Its disadvantage lies in the fact that It takes longer to set. Store in a well stoppered bottle.

Fructose (Levulose) Syrup (RI 1.47) Recommended as a temporary mountant and for special techniques.  Fructose 75g  Distilled water 25ml Glycerin Jelly (RI 1.47) An excellent routine mountant for fat stains.  Gellitin 10g  Distilled water 60ml  Glycerin 70ml  Phenol 0.25g The gelatin causes this medium to solidify and it must be melted before use.

This is easily done by placing it in the oven or a water bath at 60 C Sometime prior to use. Do not shake or stir the medium during melting as this will produce bubbles that are difficult to remove.

Highman`s Medium (RI 1.52)
This medium is recommended for use with the metachromatic dyes especially methyl violet.  Gum Arabic 20g  Sucrose 20g  Potassium acetate 20g  Sodium merthiolate 10ml  Distilled water 40ml This medium remains fluid in storage and hardens by evaporation. Store in a well stoppered bottle.

RESINOUS MOUNTING MEDIA

Resinous mountants are either natural or synthetic resins dissolved in solvents such as benzene, toluene or xylene.

Canada balsam

It is very expensive natural resin.this is prepared by collecting the resin exuded by Abies Balsamica (the Balsam fir) and dilute it in the solvent eg, xylene.

Canada balsam

As canada balsam does not mix with water mounting in it implies the use of sequence of dehydration,starting with
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Low grade alcohols followed by High grade alcohol Absolute alcohol Mixed cleaning agent plus alcohol Cleaning agent Cleaning agent mixed with xylene Pure xylene and Balsam dissolved in xylene.

toluene or benzene could be used instead of xylene ,but these solvent are equally toxic and dangerous. Development of some synthetic is substitutes for balsam doesn't solve the problem. they are equally expensive, that need the same steps and use the same toxic solvents. There are less toxic and less dangerous substances but they are expensive.

Canada balsam has an RI of1.52 very near of the glass slides and cover slips

The mounting media must always have an RI higher than the mounted sample.

Canada balsam (RI 1.52) This may be purchased as a readymade solution of 60% resin by weight in xylene or in the dry form.  If it is obtained in the dry form, it is made up as a 55 – 70% solution in xylene.  A few chips of marble in the bottom of the bottle will help reduce acidity. Coverbond (RI 1.53) This is Harleco Synthetic resin (H.S.R.) dissolved as a 60% solution by weight in toluene.it may be purchased s dry resin and may be dissolved in toluene or xylene.

DePeX (RI 1.52)

Gurr`s Neutral Mounting Medium (RI 1.51)

Gurr`s DePeX is a polysterene resin dissolved as a 20% solution in xylene. Because this mountant tends to shrink on hardening, it should be added to excess. Overflow is easily removed with a scalpel after drying is complete.

This is a mixture of coumarone and other resins as 76% solution in cineol. This mountant is a rather viscous solution. Histoclad (RI 1.54) Clay Adam’s Histoclad is a 60% solution of a synthetic resin in toluene.

Permount (RI 1.528)

Fisher’s Permount is a 60% solution of naphthalene polymer in toluene.

Pro-Texx (RI 1.495)

Technicon Resin (RI 1.62)

Pro-Texx from Lerner Laboratories is a mounting medium of neutral pH and with an antioxidant additive to preserve stain quality. It is soluble in both toluene and xylene.

UVinert (RI 1.517)

This mountant from the Technicon Corporation is a 60% solution of couramoneindene polymer in benzene and xylene in equal parts. It has a tendency to form bubbles on hardening due to high volatility of benzene. A patented mounting medium that is non fluorescent from Gurr.

Properties Of Good Mounted Sections
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Present a neat appearance. There should be no excess mountant on or around the coverglass. There should be no bubbles under the cover glass.

Usual causes of messy slides are
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Too much mountant Too thick a mountant that has not been spread to the edges of the cover glasse If the mountant is too thick it can be diluted with either toluene or xylene.

Coverglasses

Glass coverslips are used to protect specimens from physical damage and to keep them from drying out when exposed to air after staining.

COVERGLASSES

Coverglasses are either square or

rectangular

Glass coverslips are used to protect specimens from physical damage They come in different sizes and widths

Cont…

They are either 22mm or 24mm wide and come in four different lengths such as, 22mm, 30mm, 40mm or 50mm

Coverglasses

Coverglasses are manufactured to a specified thickness to complement the optical specifications of microscope objectives lenses(the recommended thickness is indicated on the barrel of the lens and is normally 0.17 mm). Various thicknesses are available (with some variation between manufacturers) and are designated

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Various thicknesses are available No. 1 (0.13 - 0.17 mm) No. 1½ (0.16 - 0.19 mm) No. 2 (0.19 - 0.25 mm)

TECHNIQUE FOR MOUNTING SECTIONS

Select the appropriately sized coverslip and place on a white paper sheet. Place a drop of mountant in the centre of the coverslip Invert the slide (section face down) over the coverslip and with one end resting on the paper sheet; gradually lower the other end until the mountant touches the section. Mountant will spread quickly over the section, between slide and coverslip.

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The slide, with coverslip attached, is then turned upright. Any trapped air is gently squeezed out whilst aligning the coverslip. The mountant is allowed to set. The time required will depend upon the particular agent used. If the result is inadequate, slides are returned to the solvent (appropriate to the mountant) to have the coverslip removed and the process repeated

Other techniques of mounting

Place the slide on a level surface, and apply a drop of mountant using the dispenser rod Hold the cover slip at a 45° angle to the surface of the slide, and allow the bottom edge to touch the drop of mountant. When the drop has spread along the edge of the slip, let go of the slip and allow the Histomount to spread slowly

Cont….

The stained section on the slide is mounted under a thin glass coverslip. The technician takes care to keep any bubbles from being trapped in the resinous mounting medium.

Cont…

Excess mounting medium may be removed while wet with a tissue, or with a razor blade when dry. Histomount will dry sufficiently to be read in 30 minutes.

LABELLING & STORAGE OF SLIDES

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After staining and mounting apply a paper label with: Name of tissue date laboratory number staining method

REFERENCES:  Theory and Practice of Histological Techniques John D Bancroft & Marilyn Gamble Fifth Edition 5. An Introduction to Histotechnology Geoffrey G. Brown, AIMLS

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