1. Rocking microtome 2. Rotary microtome - 3. Sliding microtome - 4. Freezing microtome - 5.Cryostat or cold microtome 6. Ultrathin microtome

MICROTOMY Flashcards by Queenie Mae

The process by which processed tissue, most commonly a __ , is trimmed and cut into uniformly thin slices or “sections” to facilitate studies under the microscope.

Microtomy
- paraffin embedded tissue

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is a skilled process that requires _ and _ combine with a __ by the experience histotechnologist/cian

Microtomy
- precision and hand - eye coordination
- delicate touch

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The basic instrument that is capable of cutting a section at a predetermined thickness by sliding the block into a cutting tool, usually a __, __, __ .

MICROTOME
- steel knife, glass or diamond blade fixed to a machine

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3 essential parts of microtome

Block holder
Knife Carrier and Knife
Pawl, Ratchet Feed Wheel and Adjustment Screws

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where the tissue is held in position

Block holder

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for actual cutting of tissue sections.

Knife Carrier and Knife

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to line up the tissue block in proper position with the knife, adjusting the proper thickness of the tissue for successive sections.

Pawl, Ratchet Feed Wheel and Adjustment Screws

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Principle of Microtome

• a spring-balanced teeth or pawl is brought into contact with, and turns a ratchet feed wheel connected to a micrometer screw, which is in turn rotated, moving the tissue block at a predetermined distance towards the knife for cutting sections at uniform thickness.

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kinds of microtomes:

Rocking microtome
Rotary microtome -
Sliding microtome -
Freezing microtome -
5.Cryostat or cold microtome
Ultrathin microtome

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– for cutting serial sections of large blocks of paraffin embedded tissues.

Rocking microtome

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for cutting paraffin embedded sections.

Rotary microtome

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for cutting celloidin embedded sections.

Sliding microtome

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-for cutting unembedded frozen sections.

Freezing microtome

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for cutting frozen sections

Cryostat or cold microtome –

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for cutting sections for Electron Microscopy.

Ultrathin microtome

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• This was invented by __ in __ , the simplest among the different types of microtomes.

Rocking (Cambridge) Microtome
- Paldwell Trefall
- 1881

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Rocking (Cambridge) Microtome consists of a __ and _. The lower arm resting on _ and a supporting column, and attached to the _, at the base of which is found the __ with feed mechanism.

heavy base and two arms
- pivots
- micrometer screw
- ratchet wheel

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• A section is thereby cut as the tissue passes to the knife edge in a slightly curved plane, in __ thickness.

Rocking (Cambridge) Microtome
- 10-12 u

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,available in two sizes, has been used to cut small and large blocks of paraffin tissues.

Cambridge rocking microtome

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This was invented by __ in _ to cut paraffin embedded tissues,
and is currently the most common type used for both routine and research laboratories, especially for sectioning paraffin-embedded tissues.

Rotary (Minot) Microtome
- Minot
- 1885-86

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__ are also now available and can be ideally used to produce ribbons for serial sections.

Electrically driven rotary microtomes

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• This was developed by Adams
in 1789

Sliding microtome

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There are two types of Sliding microtome

Base-Sledge Microtome
StandardSlidingMicrotome

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, consists of two movable pillars holding the adjustable knife clamps, allowing the knife to be set at an angle for cutting celloidin sections

Base-Sledge Microtome

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is favored in laboratories where very hard tissue or large blocks are usually sectioned.

Base-Sledge Microtome

theblockremainsstationarywhiletheknifeismovedbackwardand forward during the process of sectioning.

StandardSlidingMicrotome

• for cutting celloidin embedded tissue blocks.

StandardSlidingMicrotome

This was invented by _ in _ . The stage for block holder is hollow and perforated around its perimeter, attached to a reinforced flexible lead pipe thru which __passes from a cylinder.

freezing microtome - Queckett - 1848 - carbon dioxide

is used to cut undehydrated thin to semi-thin sections of fresh, frozen tissues especially in instances when rapid diagnosis is required, when - histological demonstration of fat is needed, when certain - neurological structures are to be studied, and when sensitive tissue constituents to be studied are damaged or destroyed by heat.

freezing microtome

is a refrigerated apparatus used for freezing the tissue into the block holder to the correct degree of hardness that allows for easier and faster sectioning

Cryostat

• It consists of a __, kept inside a cold chamber which has been maintained at a temperature between __ (average is _ ) by an adjustable thermostat, capable of freezing fresh tissues within _, and cutting sections of__

Cryostat - rotary microtome - -5° to -30°C - -20°C - 2-3 minutes - 4 μ

provides a means of preparing thin sections of fresh frozen tissues especially for __ or _

cryostat - fluorescent antibody staining techniques or histochemical enzyme studies.

• It is most commonly used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis

cryostat

an equipped with a glass or gem grade diamond knife is used to cut very thin sections (typically _ ) of tissue embedded in _.

ultrathin microtome - 60 to 100 nanometer - epoxy resin

Sections are stained with an aqueous solution of an appropriate heavy metal salt and examined with a _.

ultrathin microtome - transmission electron microscope (TEM)

microtome knives

plane concave biconcave plane wedge

Knives for EM

Glassknives • Diamond knives

– Used for trimming and semi-thin sectioning of tissue block

Glassknives

– Used to cut any type of resin block – Brittle and expensive, but very durable

Diamond knives

The bevel angle formed between the cutting edges normally about ___ to __

27° to 32°

– Removal of gross nicks to remove blemishes, grinding the cutting edge of the knife on a stone (__)

Honing (HEEL TO TOE) - carborundum

– Types of hones

* Belgium yellow Arkansas Fine carborundum

– for manual sharpening (best result)

Belgium yellow

– gives more polishing effect

Arkansas

– much coarser; Used only for badly nicked knives

Fine carborundum

– The process whereby the “ __” formed during honing is removed and cutting edge of the knife is polished

Stropping (TOE TO HEEL) - burr

_ is a process whereby tissues are cut into uniformly thin slices or "sections" with the aid of a microtome, to facilitate the studies under the microscope.

Sectioning

Three general types of tissue sections:

1. PARAFFIN SECTIONS 2. CELLOIDIN SECTIONS 3. FROZEN SECTIONS

- for paraffin embedded tissue blocks which may be cut by rocking and rotary microtome

PARAFFIN SECTIONS

- for celloidin embedded tissues which are usually cut by means of the sliding microtome.

CELLOIDIN SECTIONS

- which may be cut from tissues that have been fixed and frozen with CO2 or for fresh or fixed tissues frozen with the cryostat.

FROZEN SECTIONS

Factors to considered in sectioning

- Cutting depend upon the type of the tissue - The size of the block - The model or the type of the microtome *** sections usually 4-6 u in thickness for routine histologic procedure **** The knife is usually tilted at 0-15° angulation on a microtome

• A section is selected for staining and picked up onto a clean slide in a _ position

Floating Out - vertical

• Slide is immersed in the water bath in a near vertical position as close as possible to the section • Whentheslidetouchesthe section, it is lifted vertically out of water and drained

floating out

Temperature of the water bath during floating out • __, approx __ lower than the melting point of the wax used for embedding the tissue

45 -50°C, approximately 6- 10°C

• substances which can be smeared on to the slides so that the sections stick well to the slides.

ADHESIVES

• choice of __ and _ will be influenced by the staining methods to be subsequently applied.

slide and adhesive

• not necessary for routine staining, provided that the slides are clean and free from grease.

adhesive

essential for methods that require exposure of sections to acids and alkalis (especially __) during staining.

Adhesive - ammoniacal silver solutions

• This can be done either by leaving the slides in a __ , by placing the slides in a __ for _, or by drying the slides on a __ at __ for __

Section can fixed to the slides - 37°C incubator overnight - wax oven at 56° to 60°C - 2 hours - hot plate - at 45° to 55°C - for 30 to 45 minutes.

• For more delicate tissues like the _ or _ , a longer drying time at lower temperature (e.g._ for _ ) is recommended to avoid splitting and cracking of the section due to excess heat.

Section can fixed to the slides - CNS tissue or brain - 37°C for 24 hours or longer

instances when sections may float from the slide and adhesives are necessary

• Urgent cryostat sections to be submitted for immunocytochemistry • Central nervous system tissues • Tissues containing blood clot • Tissues which have been decalcified • When sections are to be subjected to high temperatures

instances when sections may float from the slide and adhesives are necessary

Common Adhesives Used

Mayer’s egg albumin Dried albumin Gelatin Starch paste plasma

Mayer’s egg albumin Made up of _,_,_,_

Glycerin, Egg white, and Thymol, crystals.

dried albumin

Sodium Chloride, Dry Albumin, and Thymol crystals

Gelatin

Gelatin, glycerol and phenol crystals dissolved in DW.

Starch paste

Powdered Starch, 2 drops of HYdrochloric acid, Thymol crystals.

plasma

readily available from outdated blood stored in blood banks.

Starch paste

Powdered Starch, 2 drops of HYdrochloric acid, Thymol crystals.

• This aqueous detergent can be purchased as a __ which is further diluted _ with _ (final dilution to 0.01%) prior to use

Poly-L-Lysine - 0.1% solution - 1:10 with distilled water

• This is widely used as a section adhesive in immunohistochemistry.

Poly-L-Lysine

are very useful in cytology, particularly for _ preparations of proteinaceous or bloody material.

APES ( 3- aminopropylthriethoxysilane) - cytospin

are better than poly-L-lysine coated slides because they can be stored for a long time without losing their ___.

APES ( 3- aminopropylthriethoxysilane) - adhesiveness

less concave sides for celloidin embedded tissue block on sliding microtome length: _

Plane- concave 25 mm

more concave paraffin sections on base- sledge rotary rocking

plane concave

for paraffin embedded section a rotary microtome length:

biconcave 120mm

for frozen section for extremely hard and tough specimen embedded in paraffin using a base sledge type or sliding microtome length:

plane wedge 100mm

MICROTOMY Flashcards

(78 cards)