Histology lectures notes Part 2 by nuhman10

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Preparation of histological specimens undergo the following steps :
(1) fixation .
(2) tissue processing :
a. dehydration .
b. clearing .
c. impregnation .
d. embedding .
(3) trimming .
(4) cutting .
(5) staining .

Once tissues are removed from the body, they undergo a process of
self-destruction or autolysis which is initiated soon after cell death by
the action of intracellular enzymes causing the breakdown of protein
and eventual liquefaction of the cell.
Autolysis is more severe in tissues which are rich in enzymes, such as
the liver, brain and kidney, and is less rapid in tissues such as elastic
fibre and collagen.
The objective of fixation is to preserve cells and tissue constituents in
as close a life-like state as possible and to allow them to undergo
further preparative procedures without change. Fixation arrests
autolysis and bacterial decomposition and stabilizes the cellular and
tissue constituents so that they withstand the subsequent stages of
tissue processing.

Fixation should also provide for the preservation of tissue substances
and proteins. Fixation is, therefore, the first step and the foundation in
a sequence of events that culminates in the final examination of a
tissue section.
It is relevant to point out that fixation in itself constitutes a major
artefact. The living cell is fluid or in a semi-fluid state, whereas
fixation produces coagulation of tissue proteins and constituents, a
necessary event to prevent their loss or diffusion during tissue
processing; the passage through hypertonic and hypotonic solutions
during tissue processing would otherwise disrupt the cells. For
example, if fresh unfixed tissues were washed for prolonged periods in
running water, severe and irreparable damage and cell lysis would
result. In contrast, if the tissues were first fixed in formalin, subsequent
immersion in water is generally harmless.

Tissue fixation
The technique of using fixatives in the preparation of cytologic,
histologic, or pathologic specimens for the purpose of maintaining the
existing form and structure of all the constituent elements.
agents employed in the preparation of histologic or pathologic
specimens for the purpose of maintaining the existing form and
structure of all of the constituent elements. Great numbers of different
agents are used; some are also decalcifying and hardening agents.
They must quickly kill and coagulate living tissue.
A large variety of fixatives is now available .
Each fixative has advantages and disadvantages, some are restrictive
while others are multipurpose
Over the years, various classifications of fixatives have been proposed,
with major divisions according to function as coagulants and non-
coagulants, or according to their chemical nature into three general

categories which include alcoholic, aldehydic and heavy metal
Factors involved in fixation
Temperature .
Size of specimens and penetration of fixative .
Changes in volume .
pH and buffers .
Osmolality .
Concentration of fixatives .
Duration of fixation .
Formulations for various fixtives
The details provided relate to commonly used fixatives. Many
variations are available and more specialised fixative solutions are not
Formaldehyde solutions
10% neutral buffer formalin (4% formaldehyde)
1 40% formaldehyde 100 ml
2 Distilled water 900 ml
3 Sodium dihydrogen orthophosphate 4 g
4 Disodium hydrogen orthophosphate (anhydrous) 6.5 g
Prepare, using quantities indicated. Fixation time: 24-72 hours.
Baker's formol-calcium (modified)
1 40% formaldehyde 100 ml
2 Distilled water 900 ml
3 10% calcium chloride 100 ml
4 7 g of cadmium chloride is sometimes added to the mixture
Prepare, using quantities indicated. Fixation time: 16-24 hours.
Formol saline
1 40% formaldehyde 100 ml
2 Sodium chloride 9 g
3 Tap water 900 ml
Prepare, using quantities indicated.
Alcoholic formaldehyde
1 40% formaldehyde 100 ml
2 95% alcohol 900 ml
3 0.5 g calcium acetate may be added to this mixture to ensure
Prepare, using quantities indicated. Fixation time: 16-24 hours.
1 Solution A
2.26% sodium dihydrogen orthophosphate 41.5 ml
2.52% sodium hydroxide 8.5 ml
Heat to 60°C-80°C in a covered container
2 Paraformaldehyde 2 g
1 Add paraformaldehyde to solution A, stirring until the mixture is
2 Filter and cool. Adjust pH to 7.2 - 7.4.
3 Prepare fresh for use (duration of fixation depends on size of
specimen and whether for light or electron microscopy).

Buffered formaldehyde-glutaraldehyde 200 mOsm38
1 Sodium dihydrogen orthophosphate 1.6 g
2 Sodium hydroxide 0.27 g
3 Distilled water 88 ml
4 40% formaldehyde 10 ml
5 50% glutaraldehyde 2 ml
Prepare, using quantities indicated. Fixation time: 16-24 hours.
Alcoholic fixatives
Carnoy's fixative
1 Absolute ethanol 60 ml
2 Chloroform 30 ml
3 Glacial acetic acid 10 ml
Prepare, using quantities indicated. Fixation time: 1-5 minutes.
1 Absolute methanol 60 ml
2 Chloroform 30 ml
3 Glacial acetic acid 10 ml
Prepare, using quantities indicated. Fixation time: 5-6 hours.
Wolman's solution
1 Absolute ethanol 95 ml
2 Glacial acetic acid 5 ml

Immerse frozen section in solution and microwave at 650 watts for 15
Acetic alcohol formalin
1 40% formaldehyde 10 ml
2 Acetic acid 5 ml
3 Ethanol 85 ml
Prepare, using quantities indicated. Fixation time: 24 hours at 4°C.
Picric acid fixatives
Rossman's fluid
1 100% ethanol saturated with picric acid 90 ml
2 Neutralised commercial formalin 10 ml
Prepare, using quantities indicated. Fix for 12-24 hours and wash very
well in 95% ethanol.
Gendre's fluid
1 90% ethanol saturated with picric acid 80 ml
2 40% formaldehyde 15 ml
3 Glacial acetic acid 5 ml
Prepare, using quantities indicated. Fixation is normally for 4 hours,
followed by washing in 80%, 95% and 100% ethanol.
Bouin's fluid
1 Saturated aqueous picric acid solution 75 ml
2 40% formaldehyde 25 ml
3 Glacial acetic acid 5 ml
1 Prepare, using quantities indicated. Fixation may vary from a few
hours to 18 hours.
2 Washing with 70% ethanol after fixation will remove most of the
yellow colour. Sections can also be washed after removal of paraffin
Mercuric fixatives
Buffered formaldehyde sublimate
1 Mercuric chloride 6 g
2 Distilled water 90 ml
3 Sodium acetate 1.25 g
4 40% formaldehyde 10 ml
Prepare, using quantities indicated. Fixation time: 16-18 hours.
Zenker's fluid
1 Distilled water 950 ml
2 Potassium dichromate 25 g
3 Mercuric chloride 50 g
4 Glacial acetic acid 50 g
Prepare, using quantities indicated. Fixation is normally for 4-24 hours
followed by an overnight wash.
Helly's fluid
1 Solution A
Distilled water 1000 ml
Potassium dichromate 25 g
Sodium sulphate 10 g
Mercuric chloride 50 g
2 Solution B
40% formaldehyde 50 ml
Add solution A to solution B immediately before use.
B5 fixative
1 Stock reagent A
Mercuric chloride 60 g
Sodium acetate 12.5 g
Distilled water l
2 Stock reagent B
10% buffered neutral formalin
To prepare a working solution mix 90 ml stock reagent A with 10 ml
stock reagent B. Fixation time: 5-8 hours.
Susa fluid
1 Distilled water 80 ml
2 40% formaldehyde 20 ml
3 Glacial acetic acid 4 ml
4 Trichloroacetic acid 2 g
5 Mercuric chloride 4.5 g
6 Sodium chloride 0.5 g
Prepare, using quantities indicated. Fixation time: 12 hours.
Principles of tissue processing
the aim of tissue processing is to embed the tissue in a solid medium
firm enough to support the tissue and give it sufficient rigidity to
enable thin sections to be cut , and yet soft enough not to damage the
knife or tissue .
The most satisfactory embedding material for routine histology is
paraffin wax. Most fixatives are aqueous-based and these are not
miscible with paraffin wax . to enable impregnation with this medium ,
the tissue must be processed . each stage must be of sufficient length to
ensure completeness .
The stages involved are :

   Dehydration : to remove fixative and water from the tissue and
    replace them with dehydrating fluid .
   Clearing : replacing the dehydrating fluid with a fluid that is
    totally miscible with both the dehydrating fluid and the
    embedding medium .
   Impregnation : replacing the clearing agent with the embedding
    medium .
   Embedding .
The first step in processing is dehydration. Water is present in tissues
in free and bound (molecular) forms. Tissues are processed to the
embedding medium by removing some or all of the free water. During
this procedure various cellular components are dissolved by
dehydrating fluids. For example, certain lipids are extracted by
anhydrous alcohols, and water soluble proteins are dissolved in the
lower aqueous alcohols
To minimise tissue distortion from diffusion currents, delicate
specimens are dehydrated in a graded ethanol series from water
through 10%-20%-50%-95%-100% ethanol.
In the paraffin wax method, following any necessary post fixation
treatment, dehydration from aqueous fixatives is usually initiated in
60%-70% ethanol, progressing through 90%-95% ethanol, then two or
three changes of absolute ethanol before proceeding to the clearing
Duration of dehydration should be kept to the minimum consistent
with the tissues being processed. Tissue blocks 1 mm thick should
receive up to 30 minutes in each alcohol, blocks 5 mm thick require up
to 90 minutes or longer in each change. Tissues may be held and
stored indefinitely in 70% ethanol without harm.
Dehydrating agents
   Ethanol is probably the most commonly used dehydrant in
Ethanol is a rapid, efficient and widely applicable dehydrant.
   Methanol is a good ethanol substitute but rarely used for routine
    processing because of its volatility, flammability and cost.
   Isopropanol was first suggested as an ethanol substitute during
    the prohibition era in the United States.
Unlike the alcohols, these reagents do not act as secondary fixatives,
and apart from solvent effects do not appear to alter tissue reactivity.

   Ethoxyethanol
   Dioxane
   Polyethylene glycols

   Acetone
   Tetrahydrofuran
   Phenol
Clearing is the transition step between dehydration and infiltration
with the embedding medium. Many dehydrants are immiscible with
paraffin wax, and a solvent (transition solvent, ante medium, or
clearant) miscible with both the dehydrant and the embedding medium
is used to facilitate the transition between dehydration and infiltration
steps. Shrinkage occurs when tissues are transferred from the
dehydrant to the transition solvent, and from transition solvent to wax
Choice of a clearing agent depends upon the following:
     The type of tissues to be processed, and the type of processing to
      be undertaken
     The processor system to be used
     Intended processing conditions such as temperature, vacuum and
     Safety factors
     Cost and convenience.
     Speedy removal of dehydrating agent .
     Ease of removal by molten paraffin wax .
     Minimal tissue damage .
Transition solvents
Toluene and xylene clear rapidly and tissues are rendered transparent,
facilitating clearing endpoint determination.

     Petroleum solvents
     Chlorinated hydrocarbons
     Chloroform
     Carbon tetrachloride
     Trichloroethane

These are colourless flammable solvents miscible with most organic
solvents and with paraffin wax.

   n-Butyl acetate
   Amyl acetate, methyl benzoate and methyl salicylate
Terpenes are isoprene polymers found in essential oils originally
derived from plants,77 though some are now synthesised.

   Cedarwood oil
   Limonene
   Terpineol
is the saturation of tissue cavities and cells by a supporting substance
which is generally, but not always, the medium in which they are
finally embedded. Tissues are infiltrated by immersion in a substance
such as a wax, which is fluid when hot and solid when cold.
Alternatively, tissues can be infiltrated with a solution of a substance
dissolved in a solvent, for example nitrocellulose in alcohol-ether,
which solidifies on evaporation of the solvent to provide a firm mass
suitable for sectioning.
Parffin wax
properties :
Paraffin wax is a polycrystalline mixture of solid hydrocarbons
produced during the refining of coal and mineral oils. It is about two
thirds the density and slightly more elastic than dried protein
The properties of paraffin wax are improved for histological purposes
by the inclusion of substances added alone or in combination to the
     improve ribboning
     increase hardness
     decrease melting point
     improve adhesion between specimen and wax
is the process by which tissues are surrounded by a medium such as
agar, gelatine, or wax which when solidified will provide sufficient
external support during sectioning.
Embedding tissues in paraffin wax
Tissues are embedded by placing them in a
mould filled with molten embedding medium
which is then allowed to solidify. Embedding requirements and
procedures are essentially the same for all waxes, and only the
technique for paraffin wax is provided here in detail. At the
completion of processing, tissues are held in clean paraffin wax which
is free of solvent and particulate matter.

                         Tissue processing
                  A modular tissue embedding centre
There are four main mould systems and associated embedding
protocols presently in use :
traditional methods using paper boats; Leuckart or Dimmock
embedding irons or metal containers; the Peel-a-way system using
disposable plastic moulds and; systems using embedding rings or
cassette-bases which become an integral part of the block and serve as
the block holder in the microtome.
Tissue processing
Embedding moulds:
(A) paper boat;
(B) metal bot mould;
(C) Dimmock embedding mould;
(D) Peel-a-way disposable mould;
(E) base mould used with embedding ring
(F) or cassette bases (G)

General Embedding Procedure
1 Open the tissue cassette, check against worksheet entry to ensure the
correct number of tissue pieces are present.
2 Select the mould, there should be sufficient room for the tissue with
allowance for at least a 2 mm surrounding margin of wax.
3 Fill the mould with paraffin wax.
4 Using warm forceps select the tissue, taking care that it does not cool
in the air; at the same time.
5 Chill the mould on the cold plate, orienting the tissue and firming it
into the wax with warmed forceps. This ensures that the correct
orientation is maintained and the tissue surface to be sectioned is kept
6 Insert the identifying label or place the labelled embedding ring or
cassette base onto the mould.
7 Cool the block on the cold plate, or carefully submerge it under
water when a thin skin has formed over the wax surface.
8 Remove the block from the mould.
9 Cross check block, label and worksheet.

Correct orientation of tissue in a mould is the most important step in
embedding. Incorrect placement of tissues may result in diagnostically
important tissue elements being missed or damaged during microtomy.
In circumstances where precise orientation is essential tissue should be
marked or agar double embedded. Usually tissues are embedded with
the surface to be cut facing down in the mould. Some general
considerations are as follows:
     elongate tissues are placed diagonally across the block
     tubular and walled specimens such as vas deferens, cysts and
      gastrointestinal tissues are embedded so as to provide transverse
      sections showing all tissue layers
     tissues with an epithelial surface such as skin, are embedded to
      provide sections in a plane at right angles to the surface (hairy or
      keratinised epithelia are oriented to face the knife diagonally)

     multiple tissue pieces are aligned across the long axis of the
      mould, and not placed at random.

Tissue orientation in the block.
(A) elongate tissues;
(B) tubular or cystic specimens;
(C) hairy skin;
(D) multiple tissue fragments.
The knife is at the lower block margin.

Processing methods and routine schedules
Tissues are usually more rapidly processed by machine than by manual
methods, although the latter can be accelerated by using microwave or
ultrasonic stimulation. For routine purposes tissues are most
conveniently processed through dehydration, clearing and infiltration
stages automatically by machine. There are two broad types of
automatic tissue processors - tissue-transfer and fluid-transfer types.
Automated tissue processing
Tissue-transfer processors :
These processors are characterised by the transfer of tissues, contained
within a basket, through a series of stationary reagents arranged in-line
or in a circular carousel plan. The rotary or carousel is the most
common model of automatic tissue processor, and was invented by
Arendt in 1909.79 It is provided with 9-10 reagent and 2-3 wax
positions, with a capacity of 30-110 cassettes depending upon the
model. Fluid agitation is achieved by vertical oscillation or rotary
motion of the tissue basket. Processing schedules are card-notched, pin
or touch pad programmed.
Processing schedules for a tissue-transfer processor. Day schedule for
urgent specimens, tissues 2 mm, fixed in Carnoy's fluid. Overnight
schedule for routine processing. Tissue blocks 2-3 mm, single load.
For a double load, immersion times should be equal. Weekend
processing: tissues are held in fixative, or preferably 70% ethanol until

         STEP           TEMPERETURE          TIME
Fixative(10%buffered            40           120
formalin)                                   minutes
Fixative(10%buffered            40           120
formalin)                                   minutes
70% alcohol(ethanol)            40            60
95% alcohol                     40            60
(ethanol)                                   minutes
Absolute alcohol                40            60
Absolute alcohol                40            60
Absolute alcohol                40            60
Xylene                          40             60
Xylene                        40            60
paraffin wax                  60            60
paraffin wax                  60            60
paraffin wax                  60            60
TOTAL TIME                                  14

Tissue-transfer processors allow maximum flexibility in the choice of
reagents and schedules that can be run on them, in particular, metal-
corrosive fixatives, a wide range of solvents, and relatively viscous
nitrocellulose solutions can all be accommodated. These machines
have a rapid turn-around time for day/night processing. In more recent
models the tissue basket is enclosed within an integrated fume hood
during agitation and transfer cycles thus overcoming the disadvantages
of earlier styles.
A tissue-transfer tissue processor with an integrated fume hood
(Shandon Citadel). Tissue cassettes are loaded into the basket on the
rotating head, which transfers tissues around the series of reagent

 1) Glass beakers / aluminum reagent containers
 2) 2 wax baths Standard tissue basket with
capacity of 80 cassettes
 3) Possibility of interrupting an automatic
process for reloading or removing cassettes for
 special applications before the end of a run
Baskets and metal cassettes should be clean and wax-free.
Tissues should not be packed too tightly in baskets so as to impede
fluid exchange.
Processors must be free of spilt fluids and wax accumulations to
reduce hazards and to ensure mechanical reliability.
Fluid levels must be higher than the specimen containers.
Timing and delay mechanism must be correctly set and checked
against the appropriate processing schedule.
A processor log should be kept in which the number of specimens
processed, processing reagent changes, temperature checks on the wax
baths and the completion of the routine maintenance schedule, is
recorded as an integral part of the laboratory quality assurance
(3)TISSUE-TRIMMING ( border molding )
The shaping of an impression material by the manipulation or action of
the tissues adjacent to the borders of an impression.
using the microtome .



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