10: Embedding, Microtomes & Microtomy

Embedding is the _ of placing cells or tissue in a __ so that thin _ can be cut using a _

technique, supporting medium, sections, microtome

Types of embedding media

Paraffin wax, celloidin, resin, plastic/polyester wax (gelatine/agar)

Tools needed for embedding

Metal and plastic moulds (various sizes), tampers, forcep, knife

1

Mould bin

2

Cold plate

3

Wax reservoir

4

Forceps warmer

5

Wax dispenser

6

Cassette-holding tank

Early form microtomoy

Free hand sectioning of fixed or fresh tissue

Types of microtomes

Rocking, base sledge, sliding, ultra, freezing (cryostat), rotary (manual or automated)

Oldest type of microtome

Rocking microtome

Base sledge microtome used for…

Large blocks, botanical histology technique for hard material, neuropatholofy and opthalmic pathology, cutting resin section

Sliding microtome used for…

cutting celloidin (embedded tissue blocks, large paraffin blocks)

Ultra microtome used for…

cutting very thin sections for use in EM studies, to look at ultra cellular structures

Ultra microtome knives

Glass (thick sections), diamond (thin sections)

Freezing microtome (cryostat) used for…

Section prepared on unfixed tissue (urgent diagnosis), rotary microtome housed in deep freezer cabinet, temp maintained -30°C to -15°C

Rotary microtome types

Manual, semi-automated, fully automated

A

Wheel handle

B

Micron selector

C

Chuck, block holder

D

Steel knife holder

E

Knife

F

Knife holder mover

G

Paraffin wax shaving holder

H

Coarse trimming lever

I

Chuck advance and back wheel

J

Chuck tilt lever

K

Angle change

L

Wheel lock

M

Knife guard

Rotary microtome advantages

Ability to cut 2–3 micron sections and cope with harder tissue,

Ideal for cutting serial sections

Tilt of knife is adjustable

Can cut resin using special holder

Manual microtome in cyostat

Rotary microtome disadvantages

Rotary motion of handle is tiring, risk of OOS (occupational overuse syndrome)

Types of knives

Steel (obsolete, tungsten tipped steal/diamond, glass, disposable (stainless steel coated)

Tungsten carbide/diamond knives

For very hard undecalcified bone, resin blocks

Tougher than steel, industrially sharpened

Glass knives

Mainly for resin blocks for light and electron microscopy, high quality knives made using special equipment, prepared just prior to use

Advantages of disposable knives

Widely used for microtomy of paraffin block, sharpening not required and is assured, easy to use, cost effective, replacement blades readily available, low and high profiles adaptable to variety of tissue and paraffintype, safe storage when not in use, safe disposal

Microtomy

Ability to cut extremely thin slices (sections) of tissue

Purpose of microtomy

Enables transparency of stained tissues and cells when viewing microscopically

Needed for microtomy

Thorough knowledge of equipment used and type of investigation required, good quality tissue (well fixed)

Routine histolofgy sections are cut at…

3–4μm thickness

Microtomy tools

Microtome, water bath (45°C), tissue paper, slide rack, forceps, disposable blades, cold plate/ice, slides ± adhesive, pencil, brush

Single full-face section

Single section

Serial section

Consecutive sections

Stepped levels section

Sections at periodic levels

Type of slide depends on…

investigation

Plain/frosted end (noncoated) slides used for…

routine histology

Coated/adhesive slides

Commercially pre-coated, charged slides

Coated slides used for…

Sections exposed to strong alkali, cryostat (frozen) sections, immunochemistry and direct immunofluorescence studies, CNS tissues, blood clots, decalcified tissues

Slide drying purpose

Slide drying in…

hot place, laboratory oven

Folds

Knife marks

Bubbles under section

Dust present

Excessive compression

Over expansion

Cause and remedy: Section ribbon curved

Knife is blunt

Change knife

Cause and remedy: Scoring or splitting of sections

Nick in knife edge, hard particles in tissue/wax

Change knife, use softening agent/remove hard particle and re-embed

Cause and remedy: Areas of tissue in block not present in section

Too vigorous trimming, incomplete trimming

Polish block, further trim block

Cause and remedy: Holes in section

Harsh or coarse trimming

Fine trim (polish) block to get rid of tough appearance

Cause and remedy: Blade hits into block and tissue is cut out

Tissue is loose in block, microtome part not clamped down, block is not secure in holder, aggressive trimming with rapid wheel rotation

Re-embed tissue, tighten clamps, re-secure block, slow down trimming