paraffin and microtome combination

Microtomy

Process by which a processed tissue, most commonly a paraffin embedded tissue, is trimmed and cut into uniformly thin slices or “sections” to facilitate studies under the microscope

Identify the parts of the microtome

• block holder

• knife carrier and knife

• pawl, ratchet feed wheel and adjustment screws

3 Essential Parts of a Microtome

Block holder

Where the tissue is held in position

Knife carrier and knife

For actual cutting of tissue sections

Pawl, ratchet feed wheel and adjustment screws

part of the microtome To line up the tissue block in proper position with the knife, adjusting the proper thickness of the tissue for successive sections

Paldwell Trefall (1881)

Rocking (Cambridge) Microtome is invented by

Rocking (Cambridge) Microtome

Simplest and oldest among the different types of microtomes; Only used to cut small and large blocks of paraffin tissues

True

T/F: for Rocking (Cambridge) Microtome, the size of the block that can be cut is limited

True

T/F: for Rocking (Cambridge) Microtome, no serial section is possible since tissues are cut in slightly curved planes

False

T/F: for Rocking (Cambridge) Microtome, serial section is possible since tissues are cut in slightly curved planes

Minot (1885-1886)

Rotary Microtome invented by _______ in

paraffin

Rotary microtome can cut _______-embedded tissues

Rotary (Minot) Microtome

Most common type used for both routine and research laboratories

Rotary (Minot) Microtome

this microtome has adjusting screws to make the tissue block parallel to the knife

Sliding Microtome

Developed by Adams in 1789

• base-sledge microtome

• standard sliding microtome

types of sliding microtomes

Base – Sledge Microtome

Consists of 2 movable pillars holding the adjustable knife clamps, allowing the knife to be set at an angle for cutting celloidin sections

base-sledge microtome

microtome For hard tissue or large blocks that are usually sectioned

Standard sliding microtome

Recommended for cutting extremely hard and rough tissue blocks; Most dangerous type of microtome due to the movable exposed knife

celloidin

Standard sliding microtome was developed for cutting __________ – embedded tissue blocks

Freezing Microtome

Used to cut undehydrated tissues in a frozen state, especially when rapid diagnosis is required

Queckett (1848)

Freezing Microtome was invented by ______ in what year?

Freezing Microtome

microtome where Histological demonstration of fat is needed; Neurological structures are to be studied and Sensitive tissue constituents are easily destroyed or damaged by heat

Cryostat or Cold Microtome

A refrigerated apparatus used in fresh tissue microtomy for freezing the tissue into the block holder to the correct degree of hardness to facilitate easier and faster sectioning

rotary microtome

Cryostat or Cold Microtome Consists of what microtome which is kept inside a cold chamber

-5⁰C to -30⁰C (20⁰C)

Cryostat or Cold Microtome is maintained at a temperature between _____ to _____ (average is ____) by an adjustable thermostat

Cryostat or Cold Microtome

microtome used in Fluorescent antibody staining techniques or histochemical enzyme studies

Ultrathin Microtome

Used for cutting sections at 0.5 micra

electron microscopy

what type of microscope is attached to the Ultrathin Microtome

osmium tetroxide; plastic

in ultrathin microtome, the spx is small and fixed in what fixative and embedded in _____

• Plane – Concave knife

• Biconcave Knife

• Plane – Wedge Knife

types of Microtome knives

Plane – Concave knife

one side of the knife is flat while the other is concave

25mm

Plane – Concave knife length

celloidin; sliding

Less concave sides are recommended for cutting __________ – embedded tissue blocks on a _________ microtome

paraffin; base – sledge, rotary or rockingkn

More concave sides are used to cut _________ sections on _________ microtome

Biconcave knife

knife With both sides concave

120mm

Biconcave knife size

Biconcave knife

knife that is recommended for cutting paraffin embedded sections on a rotary microtome

Plane-Wedge Knife

knife that has both sides straight

100mm

Plane-Wedge Knife length

Plane-Wedge Knife

Recommended for frozen sections or for cutting extremely hard and tough specimens embedded in paraffin blocks, using a base – sledge type or sliding microtome

True

T/F: There is a cutting facet (bevel) found on the tapered edge of all knives, the sides which are more acutely inclined towards each other, then the side proper, forming the actual cutting edge of all knives

BEVEL ANGLE

Angle formed between the cutting edges is known as the

27⁰ to 32⁰

normal BEVEL ANGLE range

steel; 2-3 microns; serrations

A good cutting edge should be made of good quality ________ and be able to cut good sections from a paraffin wax block about _____________ thick, without any ________ noted on examination

15⁰

The perfect and optimum cutting angle is obtained when the sides of the wedge knife are inclined at an angle of about ____

tears or striae

Jagged edges – produce ____ or ____ in tissue sections

wiped clean

Before doing the honing, surface of the hone must be

– Mineral and Clove oil

– Xylene

– Liquid paraffin (obsolete)

– Soapy water

the surface of the hone is covered with a thin film of any of the following for lubrication:

Honing

General term involving the removal of gross nicks on the knife edge to remove blemishes and then grinding the cutting edge of the knife on a stone to acquire an even edge

Coarse honing

removal of gross nicks on the knife edge to remove blemishes

Honing Proper

grinding the cutting edge of the knife on a stone to acquire an even edge

blade, knife carrier, handle, bevel

parts of the knife used in honing

Honing

refers to removal of fine nicks

Heel to toe

motion of honing

Stropping

refers to sharpening the knife which is free of nicks

Toe to Heel (reverse)

Stropping motion

carborundum

Honing Makes use of a hone, a natural sharpening stone or hard grinding surface (____________) which serves to remove nicks and irregularities on the knife edges

Belgium Yellow

For manual sharpening when cutting edge has been rendered blunt or nicked. This type usually gives the best result

Arkansas

Gives more polishing effect than the Belgium Yellow

Fine Carborundum

Much courser than the first two types and is used only for badly nicked knives followed by either one of the first two knife sharpeners

Plate Glass Honing

A flat circular glass plate with finely powdered aluminum oxide made into paste with water (used as an abrasive)

Diamantine

what is used for Plate Glass Honing as the final polishing

Automatic Hone

Honing that is Fast; becoming indispensable in histopath laboratories; Time saving and fairly easy to manipulate; Consists of glass disc or wheel driver by an electric motor

• The hone should be long enough to allow the whole length of the knife edge to be sharpened (8x3 inches)

• The hone should be lubricated with warm soapy water or fine oil before using

• The pressure should be gentle and steady

• The amount of strokes should be 20-30 times in each direction

• The hone should be cleaned before, during and after use

• A black film that develops in the hone usually is imparted by the knife that is being sharpened and should be brushed out with a good nailbrush in running water

Precautions during honing

Stropping

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

Stropping

polish and sharpen the cutting edge

Honing

remove irregularities from the knife

True

T/F: If the knife has become dull and blunt but is free from nicks, it is usually only necessary to strop it

False

T/F: If the knife has become dull and blunt but is free from nicks, it is usually only necessary to hone it

paddle strop

It is made up of the best quality horse leather, firmly attached to a solid back, in order to prevent sagging

• The knife should always be wiped clean before and after use

• The knife edge should be oiled or greased after use to prevent from rusting

• The pressure should also be light and gentle

• Too much oil will making stropping slippery and will render unsatisfactory results

• The knife should be flushed with xylene

Precautions observed in stropping

Disposable blades

has Sharp cutting edge that can cut 2-4µ thick sections with ease

Glass Knives

Generally used for trimming and semi – thin sectioning of tissue blocks for EM

Diamond Knives

Used to cut any type of resin block for EM; Brittle and expensive, but very durable and the cutting edge must be kept clean to make it cut longer and to avoid damage during sectioning

• Waterbath

• Drying oven or hot plate

• Forceps (fine pointed or curved) and squirrel hair brush

• Clean slides

• Ice tray

• Pencil

Other equipment used in microtomy

Sectioning

a process whereby tissues are cut into uniformly thin slices or “sections” with the aid of a machine, to facilitate the studies under the microscope

Coarse and Fine Trimming

types of Trimming

15mm

for fine trimming, Setting the thickness adjuster at

type of tissue, size of block and model of the microtome

Cutting rate depends on

4-6 microns

Sections are cut between ___________ in thickness for routine procedures

ten

Sections are removed in ribbons of ____ to allow easy location of serial sections

10

Block is faced and one ribbon of ____ sections is cut

first and last section

what sections of the ribbon are taken for the first two levels

8 sections in between

in one ribbon, what sections are discarded

Incomplete sections

generally, what sections are discarded

• camel hair brush

• pair of forceps

• fingers

Complete sections are picked up at once using

exhaling gently

Tissue that tend to crumble or do not form a smooth flat surface can be sectioned with ease, by ____________ into the block surface while the section is being cut slowly to reduce the effects of static electricity

45-50C; 6-10C

The sections are floated out on a water bath set at ______, ______ lower than the melting point of the wax

30 seconds

Sections should not be left on the water bath for a long time (max. of how many seconds); to avoid undue expansion and distortion of tissue

10-12 inches

diameter of a floating out batch

3-4 inches

depth of a floating out batch

Black

color of the inside of a floating out bath

½-1 cm

Bath should be filled with water to within ______ from the top

emptied and thoroughly wiped clean

what should be done to the floating out bath after use

Adhesive mixtures

Reduces the surface tension thereby producing closer capillary adhesion of the sections to the slides

Protein

Adhesive mixtures are made up of what solutions

Mayer’s egg albumin glycerol

Most popular adhesive mixture

50mL of white fresh egg and 50mL of glycerol

Mayer’s egg albumin glycerol is composed of