Mounting and Labelling- FINALS L13

MOUNTING MEDIUM

• It is a syrupy fluid applied between the section and the coverslip after staining.

• Mounting a tissue specimen is essential for preserving the specimen during storage as well as for enhancing imaging quality during microscopy.

PURPOSES OF MOUNTING MEDIUM

1. To promote adhesion of the specimen

2. To promote adhesion of the glass slide

3. To prevent movement of the specimen

4. To preserve the specimen

IF AN UMNOUNTED 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.

CHARACTERISTICS OF A GOOD MOUNTING MEDIUM

• Refractive index should be as near as possible to that of the glass which is 1.518

• RI-ratio of speed of light in air and speed of light in a specific medium.

• It should freely miscible with xylene or toluene

• It should not dry quickly

• It should not crack or produce artefactual granularity on the slide upon drying

• It should not dissolve out or fade tissue sections

• It should not cause shrinkage and distortion of the tissue

• It should not leach out any stain or affect staining

• It should not change in color and pH

• It should set hard thereby producing permanent section

MOUNTING MEDIUMS

• Aqueous media

• Resinous media

AQUEOUS MEDIA

They are designed to mount water miscible preparations directly from water where the stained is removed or decolorized with alcohol or xylene.

AQUEOUS MEDIA IS MADE- UP OF THE FOLLOWING

• Substance that will solidify the medium

• Substance that will prevent cracking & drying of the preparation subs. That will increase refractive index

AQUEOUS MEDIA PRESERVATIVE

Merthiolate

TYPES OF AQUEOUS MEDIA

• Water

• Glycerin

  • Glycerin Jelly

• Farrant’s medium

• Karo corn syrup

• Mineral oil

• Von apathy’s gum syrup

• Levulose (Fructose)

• Brun’s fluid

WATER

Has low RI, moderately transparent, evap. easily, good only for temporary mounting

GLYCERIN

May be used as preservative, has high refractive index, last for a few minutes, RI 1.46

GLYCERIN JELLY

RI 1.47

FARRANT’S MEDIUM

RI 1.43

KARO CORN SYRUP

RI 1.484 to 1.486 at 20*C

MINERAL OIL

• Useful temporary mounting media for romanowsky stained smear

• RI 1.47-1.48, common value is 1.467 at 20*C

VON APATHY’S GUM SYRUP

RI 1.52, made of pure gum arabic, pure cane sugar or sucrose, dist. water, and thymol.

LEVULOSE (FRUCTOSE)

RI: 1.5

BRUN’S FLUID

• Made of glucose, glycerin, spirit of camphor, and distilled water.

• Recommended for mounting frozen sections from water

RESINOUS MEDIA

Canada Balsam

CANADA BALSAM

• Natural resin extracted from the Canadian Tree – Abus balmasea/ Abies balsamea

• Transparent and colorless

• Sets to a hard consistency

• Excellent mounting medium that ensures long lasting preservation and clarity for specimen

TWO CATEGORIES OF MOUNTING MEDIA

1. Organic (hydrophobic)

2. Aqueous (hydrophilic)

ORGANIC MOUNTING MEDIA

Can only be used for enzymatic labels where the precipitate formed between the enzyme and the chromogen is not soluble in the organic solvents used during mounting of the tissue (e.g. diaminobenzidine, DAB).

AQUEOUS MOUNTING MEDIA

Are generally suitable for all enzymatic label/chromogen combinations and fluorescent labels.

SYNTHETIC BRANDS SUPERIOR THAN CANADA BALSAM

1. DPX of Kirkpatrick and Lendrum

2. Clarite X

3. Permount, Clearmount, Histosec, Harleco synthetic resin, Entallan

4. XAM

5. Eukitt

DPX OF KIRKPARTICK AND LENDRUM

• Resin “distrene 80” and tricresyl phosphate or dibutyl phthalate

• Shrink somewhat on drying

• Good mounting medium

• RI 1.52

CLARITE X

• One of the widely used mounting media

• Synthetic resin

• RI 1.544

XAM

• RI 1.52

• Obsolete

EUKITT

• It is an adhesive and specimen preservative

• It contain 5% Acrylic Resin and 55% Xylene

PROPERTIES OF EUKITT

• Fast drying with minimal air bubbles

• RI close to the glass (1.510)

• Chemically neutral, little sensitivity to water

• No reaction to staining

• Non-yellowing, unaffected by cold and UV

• Stays optically clear for over 10 years

• Does not cryztallize

FINISHED SLIDES

• Easily reworked by immersing previously mounted slide in xylene to remove coverslip.

• The slide may then be incubated at 37*C for 12-24 Hrs. after mounting, to harden the medium.

• Setting may be hastened in a hot oven at 50*C for 2 hrs.

AFTER IMMUNOCHEMICAL STAINING

Mounting media are used to adhere a coverslip to a tissue section or cell smear. Mounting media are important to protect the specimen and staining from physical damage. Mounting media also play an important role in improving the clarity and contrast of an image during microscopy.

RINGING

• Sealing the margin of the coverslip

• Krognig cement is made of 2 parts paraffin wax with 4 9 parts of powdered colophonium resin

COVERSLIPPING

• The stained section on the slide is covered with a thin piece of glass to protect the tissue from scratched, to provide better optical quality for viewing under the microscope, and to preserve the tissue section for years to come.

• Cover slips are identified by number which pertain to the thickness

• As thickness increases, transparency is reduced.

• Cover glasses come in different sizes, namely 18x18 mm, 20x20 mm, 22x22 mm, & 25x25 mm.

• The choice of cover glass depends on the size of the tissue to be mounted.

LABELLING

• Slide with frosted edge, use pencil to label the specimen

• Must be written clearly.

• The labels may be written, etched, or embossed on each slide