Adhesives, Mounting and Labeling

• Section cutting


  Process of cutting a processed tissue into uniformly thin slices (sections) using a microtome to facilitate studies under the microscope

  
• Conventional tissue processing steps

  • Fixation
  • Decalcification
  • Dehydration
  • Clearing
  • Impregnation/Infiltration
  • Embedding
  • Trimming
  • Section-cutting (microtomy)
  • Staining
  • Mounting
  • Labeling
• Trimming


  1. Coarse trimming

  2. Fine trimming
• Coarse trimming


  Sides, tips, and bottom of the tissue are trimmed using a knife or a blade to form a truncated pyramid/4-sided prism

  
• Fine trimming


  Block is placed in the microtome, setting the adjuster at 15mm or by advancing the block using the coarse feed mechanism, where the surface is trimmed away until the entire tissue surface has been exposed

  
• Sectioning/cutting


  Process wherein a processed tissue is cut into uniformly thin slices (sections) using a microtome to facilitate studies under the microscope

  
• Methods of picking up the section

  • Index finger
  • Camel hairbrush
  • Spatula
  • Flat-bladed forceps
• Types of sections and their usual thickness

  • Paraffin section: 4-6 micra
  • Celloidin section: 10-15 micra
  • Frozen section: 10 micra
  • Ultrathin section: Semi thin: 0.5 - 1 micron, Ultra thin: 500-1200 angstrom or 50-120 nm
• Fishing out


  1. Sections are floated out on a water bath set at 45°C to 50°C to flatten and prepare them for mounting onto a slide

  2. Section is then selected and picked up onto a clean slide in a vertical position
• Methods of drying slides

  • Wax oven at 56°C - 2 hours
  • Incubator at 37°C - overnight
  • Hot plate at 45-55°C - 45 min
  • Blower type slide dryer at 50-55°C - 20-30 minutes
  • Carefully holding the slide section upwards, above a Bunsen flame, adding mounting medium until the wax melts (for delicate tissues)
• Adhesives


  Used to promote adhesion of sections to slides, spread thinly and evenly on a clean grease-free slide
• Types of adhesives


  • Mayer's egg albumin
  • Dried albumin
  • Starch paste
  • Plasma
  • Gelatin 1%
  • Gelatin-formaldehyde mixture
  • Poly-L-Lysin
  • APES (3-aminopropylthriethoxysilane)
• Faults, reasons, and remedies

  • Brittle or hard tissue
  • Clearing agent turns milky
  • Tissue smells of clearing agent
  • Tissue is opaque, section-cutting is difficult
  • Tissue is soft when block is trimmed
  • Airholes found on tissue during trimming
  • Wax appears crystalline
  • Paraffin block is moist and crumbles
  • Sections fail to form ribbons
  • Sections roll up on cutting
  • Ribbon is curved, crooked or uneven
  • Sections are compressed, wrinkled or jammed
  • Sections are squashed
  • A hole is formed in the section
  • Sections of unequal thickness
  • Tissue shrinks away from wax
  • Sections adhere to the knife or other parts
  • Ribbon is split or has vertical scratches
  • Sections are lifted from the knife
  • Resistance is felt on the lower part of the section
  • Horizontal or parallel lines or furrows across the section
  • Section cut is sometimes thin, sometimes thick
  • Frozen tissue crumbles and comes off the block holder
  • Frozen tissue chips into fragments

  
• Mounting


  Protects the stained section, facilitates handling and storage, prevents bleaching or deterioration, preserves the slides for permanent keeping, and prevents distortion of the image during microscope examination
• Aqueous mounting media


  • Water
  • Glycerin
  • Glycerin jelly
  • Apathy's medium
  • Farrant's medium
  • Brun's fluid
• Slides used must always be grease and dust-free and stored and handled correctly
• en cut


  Tissue is frozen too hard
• Warming frozen tissue


  Warm the tissue with the fingers
• MOUNTING


  • Protects the stained section from getting scratched
  • Facilitate easy handling and storage of the slides
  • Prevent bleaching or deterioration due to oxidation
  • Preserves the slides for permanent keeping
  • Prevent the distortion of image during microscope examination
• Aqueous Mounting Media


  • Water
  • Glycerin
• Glycerin Jelly


  Refractive Index 1.47, contains gelatin, glycerol, distilled water and phenol crystals
• Apathy's Medium


  Refractive Index 1.52, contains pure gum arabic, cane sugar or sucrose, distilled water, thymol crystals
• Farrant's Medium


  Refractive Index 1.43, contains gum arabic, distilled water, glycerol and Sodium merthiolate
• Brun's fluid


  Contains glucose, glycerine, spirits of camphor, Distilled water
• MOUNTING


  Process of putting cover sip on the stained tissue using mounting medium to stick the cover slip on the side. It is done to facilitate proper handling and to prevent damage to the section.
• Mounting Media


  • Solidify the medium
  • Prevent cracking & drying of the preparation
  • Raise the refractive index
• Mounting Media


  • Gelatin
  • Glycerin jelly
  • Gum Arabic
  • Glycerol
  • Sugar
• Resinous Mounting Media


  • Canada Balsam (RI 1.524)
  • D.P.X. (RI 1.532)
  • XAM (RI 1.52)
  • Clarite (RI 1.544)
  • Permount (Fisher Scientific)
  • HSR (Harleco Synthetic Resin)
  • Clearmount (Gurr)
• Characteristics of a good mounting medium

  • Colorless and transparent
  • Freely miscible with xylene and toluene
  • Should not dry to a non-stick consistency and harden relatively quickly
  • Protect the section from physical damage and chemical activity
  • Resistant to contamination
  • Should not cause shrinkage and distortion of tissues
  • Should not leach out any stain or affect staining
  • Should not change in color or pH
  • Compatible with the adhesive in use
  • Should set without crystallizing, cracking or shrinking
• RINGING


  Process of sealing the margins of the cover slip to prevent escape of fluid/semi-fluid mounts and evaporation of the mountant. It is done to immobilize the cover slip and to avoid sticking of the slides upon storage
• Ringing


  A liquid preparation sealed well with nail polish could last for months. Paraffin wax may be applied with a ringing iron and is satisfactory as a temporary ringing agent.
• LABELING


  Process of indicating the year and specimen number on one end of the prepared slide for proper identification
• Information on slide label

  • Year, last 2 digits
  • Specimen number
  • Pencil is used
• Slides should be properly labeled with an identifying case number on the side of the mounted coverslip. As a general rule, a paper label bearing the patient's name, section number, and preferably the staining method used, is attached to the slide for proper identification, while also avoiding any damages to the sections caused by wiping the "wrong" side of the slide.
• The quality of the sections and quality of staining produced by the histopathology laboratory must be checked before issuing them to the pathologist.
• Handling broken slides


  1. Mounting a broken slide onto another clean xylene-moist slide with a drop of mounting media (Clarite or permount) may be sufficient for immediate examination while a new section is being cut and stained. If an important slide is broken and replacement is not available, the section -if still intact- may be transferred to another slide.

  2. If the slide is broken only on the end far from the tissue, re-affix the broken fragment with a cellophane tape.

  3. If the slide is broken in several pieces and if enough pieces are present: 1. Reconstitute the pieces on a blank slide using mounting medium as the adhesive, 2. Wipe them gingerly with xylene, 3. Dry the slide flat on a hot plate or in a 37°C oven for several hours or overnight.

  4. If the cover slip has also been fractured, it is necessary to loosen the broken pieces with xylene and apply a new cover slip.
• Disengaging stuck slides


  Soak the slides in a dish of xylene to dissolve the sticky mounting medium. When the slides are separated, clean and replace the cover slip as needed.
• FROZEN SECTION


  • Rapid diagnosis during surgery
  • Diagnostic and research enzyme histochemistry
  • Diagnostic and research demonstration of soluble substances such as lipids and carbohydrates
  • Immunofluorescent and immunohistochemical staining
  • Some specialized silver stains, particularly in neuropathology
• Methods of Preparing Frozen Sections

  • Cold Knife Procedure
  • Cryostat Procedure
• Cold Knife Procedure


  Different temperature is employed between the tissue and the knife, Carbon dioxide gas is the commonly used freezing agent, DEW LINE: point @ which sections may be cut @ micra thickness