Mounting Media and Adhesives

56問 • 2年前

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It is the last step in tissue processing that results in a permanent histological preparation

Mounting

Describe the process done in Drying

After cutting: Sections are floated out on a water-bath, Bubbles accumulating under the ribbon may be removed with a smooth teasing needle or by pulling the ribbon very gently When sections chosen have flatten out: Numbered slide is immersed in the water bath and; Section is fished out and drained After draining: Sections are fixed to the slides – done by (1) leaving the slides in a 37C incubator overnight, or (2) placing the slides in a wax oven at 56 to 60C for 2 hours, or (3) placed at hot plate at 45 to 55C for 30 to 45 mins

What is another alternative method to drying?

Use of adhesive

Substances smeared onto the slides so that the sections stick well to the slide (Slide should be grease and dust-free)

Adhesives

What is the purpose of using adhesives?

To stick the sections to the slide

What are the common type of Adhesives?

Mayer's Egg Albumin Dried Albumin Gelatin Gelatin-formaldehyde mixture Starch Paste Plasma Poly-L-Lysine APES (3-Aminopropylthriethoxysilane)

Most commonly used adhesive in histopathology because it is easy to make, convenient, and relatively inexpensive

Mayer's Egg Albumin

Composition of Mayer's Egg Albumin

50cc Egg white 50cc Glycerin Water

What are the disadvantages of Mayer's Egg Albumin?

→ retain some of the stains → gives a dirty background in sections

True/False: Excess drop should be wiped off, too little adhesive ---> will not stick well

True

Composition of Dried Albumin

5g Albumin 5g NaCl 100cc Distilled water

Composition of Gelatin (1%)

1g Gelatin 100mL water 15mL glycerol Phenol crystal

Composition of Gelatin-formaldehyde mixture

5 ml 1% Gelatin 5 ml 2% formaldehyde

Composition of Starch Paste

1g powdered starch 30 ml water 2 drops of HCl

This adhesive is readily available from outdated blood stored in blood banks dispensed into sterile tube of 0.5ml each

Plasma

Widely used as a section adhesive in immunohistochemistry

Poly-L-Lysine

Must be used within a few days after preparation ---- its effectiveness as an adhesive decrease in time

Poly-L-Lysine

Useful in cytology especially for cytospin preparations of proteinaceous or blood material

APES (3-Aminopropylthriethoxysilane)

Why is APES-coated slides better than Poly-L-Lysine coated slides?

Because they can be stored for a long time without losing their adhesiveness

It is usually a syrupy fluid applied between the section and the coverslip after staining, setting the section firmly– preventing the movement of the coverslip

Mounting medium

-The mounting medium must have a refractive index ______ to that of the glass slide and tissue -The mounting media must have an RI________ than the mounted sample to impart more transparency

close Higher

-It will enhance specimen detail (e.g. choosing a RI similar to glass will make the glass invisible -Governs the contrast between the cellular detail and the background

Refractive index

What are the advantages of Mounting Medium?

1) Mounting medium protects section from getting scratched 2) Facilitate easy handling at storage 3) Prevent bleaching or deterioration due to oxidation– preserving the slides for permanent keeping

True/False: Unmounted stained section will result in very little microscopic detail can be appreciated

True

What are the characteristics of a Good Mounting Medium?

1) Avoid tissue distortion 2) Miscible with xylene and toluene 3) Should not dry quickly 4) Should not crack or produce artefactual granularity on the slide upon drying 5) Should not dissolve out or fade tissue sections 6) Should not cause shrinkage and tissue distortions 7) Should not leach out any stain or affect staining 8) Should not change color or pH 9) Should set hard, producing permanent mounting of sections

-Excess mounting medium in sections must be wiped off with fine cloth moistened with ______. -Basic aniline dyes should be mounted with non-acid containing mountants (basic)

xylene

What are the two main groups of Mounting Media?

Aqueous Mounting Media Resinous Mounting Media

This type of Mounting media is designed to mount water-miscible preparations directly from water in cases where the stain is removed or decolorized with alcohol or xylene.

Aqueous Mounting Media

Aqueous Mounting Media are usually made up of what?

Gelatin, glycerin jelly or gum Arabic Glycerol Sugar

It is used to solidify aqueous mounting media

Gelatin, glycerin jelly or gum Arabic

What is used to prevent cracking and drying of the preparation in Aqueous Mounting Media?

Glycerol

What is used to increase Refractive Index in Aqueous Mounting Media?

Sugar

-Has low refractive index, moderately transparent and evaporates easily -Good for temporary mounting

Water

-High RI -Provides greater visibility if slightly diluted with water -Mountant for fat stains -Inexpensive and not poisonous

Glycerin

What is a variety of Glycerin for Aqueous Mounting Media?

Phosphate Buffered Glycerin

This variety of Glycerin is used to mount sections for Immunofluorescence

Phosphate Buffered Glycerin

What are the examples of Glycerin Media?

Glycerin jelly Farrant's medium Brun's fluid Apathy's medium

This glycerin media is recommended for mounting frozen sections from water

Brun's fluid

-This glycerin media is used for methylene blue-stained nerve preparations and as a general purpose aqueous mountant -Also used for fluorescent microscopy

Apathy's medium

What is used in Farrant's medium?

Sodium merthiolate

What is an alternative for Sodium merthiolate in Farrant/s medium?

Arsenic trioxide

-This glycerin media is used when dehydration and clearing with xylene cannot be made (as in fat stains) -Stains tends to fade out

Glycerin jelly

What is used as an alternative to glycerin jelly as mountant in immunofluorescence?

Polyvinyl alcohol

This type of Mounting media are used for preparations that have been dehydrated and cleared in xylene or toluene

Resinous Mounting Media

What are the different resinous mediums under Resinous Mounting Media?

Canada Balsam DPX (Dibutyl Phthlate and Xylene) XAM CLARITE (Clarite X)

-Synthetic resin soluble in xylene -Preferred than DPX

CLARITE (Clarite X)

-Synthetic resin mixture in xylene -Available in pale yellow or colorless solution -Preserve stains well

XAM

-Resinous medium, for small tissue sections -For standard stains – well preserved

DPX (Dibutyl Phthlate and Xylene)

-Natural resin extract from Canadian tree (Abus Balsamea) -Dissolved in xylene in an incubator at 37C or paraffin oven at 548C -Transparent, almost colorless oleoresin that adheres firmly to glass and sets to a hard consistency without granulation -Recommended for whole mounts and thick sections

Canada Balsam

After adding the mounting medium, the stained section on the slide must be covered with a thin piece of plastic or glass (cover slip), what do you call this process?

Cover slipping

What are the purpose of cover slipping?

1) Protect the tissue from getting scratched 2) Provide better optical quality for viewing under microscope 3) Preserve the tissue section for years to come

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

Ringing

Give an example of ringing medium

Nail polish

Give an example of temporary ringing agent

Paraffin wax applied with a ringing iron

What is the Ringing media used in Ringing process?

Kronig cement

What is the purpose of Ringing?

Immobilize the cover slip Prevent stick of the slide upon storage

Translation(Pre-finals)

MAHATHIR ALPHA · 55問 · 2年前

Translation(Pre-finals)