Microtechniques and TISSUE PROCESSING (HISTOTECHNIQUES), Study Guides, Projects, Research of Microbiology

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Microtechniques are a group of processes that can beused

for

the

preparation

of

different

types

of

biological samples to produce microscopic slides thatcan be viewed under the microscope. This includesthe following techniques:

Tissue Processing

Smears

Whole mount

1-OBTAINING THE SPECIMEN

This differs according to the type of

the specimen and the

type of the living organism from which the specimen will betaken. The specimen can be taken by: 

-Biopsy (taking a small piece of tissue from alive organism by special machine).



-Autopsy (taking the specimen soon after death by the same way of biopsy)



-Surgery (in case of surgical operations of some animals or human)



-Dissection (in case of different types of animals)

DISSECTION Animals must be anesthetized before dissection by using a

suitable

dose

of

an

anaesthetic

substances

that

can

anesthetized

by

not

kill

the

animal.

Among

these

substances are:



Ethyl

ether

for

insects

arachnids

and

most

terrestrial

vertebrates



Chloroform

for

some

insects

and

most

terrestrial

vertebrates



Chloroform + Acetone for most invertebrates



Methanol for marine invertebrates and mollusca



Barbiturate for large vertebrate animals



-Abdominal dissection should start by making a small opening in the skin atthe top of the cloacal or anus opening.



-A

piece

from

the

wanted

organ

should be cut and removed from thebody of the animal then washed fromblood in a physiological saline solution(0.85% NaCl).



The piece of tissue should be rapidly placed in a selected fixative or left in aclean

physiological

solution

till

the

end of the dissection then transferredto the fixative.

2-SPECIMEN ACCESSIONING

Tissue

specimens

received

are

accessioned

by

giving

them

a

number

that

will

identify

each

specimen.

This

number

showed

indicates the type of the tissue, thetype

of

treatment

including

the

dose and the route and duration ofinjection, types of fixative that willbe

used

and

other

important

information

about

the

specimen

The number should be written on awhite sticker by a pencil and fixedon

the

container

in

which

the

specimen will be placed.



It is important to realise that a fixative will initially

produce a number of changes to the tissues in what is usuallyan aqueous environment. These will include shrinkage, swellingand hardening of

various components. Despite these initial

effects tissues will undergo further changes during processingwhen they are placed in a non-aqueous environment. 

For example fixation in 10% buffered formalin initially

causes slight swelling of

tissue specimens. During processing

however the specimen may shrink 20% - 30% of its volume.

TYPES OF FIXATION

Fixation of tissues can be achieved by chemical or physicalmeans

.

Physical methods; include heating, micro-waving and freezedrying.

Chemical fixation; achieved by immersing the specimen in thefixative (

immersion fixation

) or, in the case of small animals or

some whole organs such as a lung, by perfusing the vascularsystem with fixative (

perfusion fixation

Fixative solutions may contain a single fixative agent dissolvedin a solvent such as water or alcohol or more commonly, a buffersolution to stabilize pH. Some popular fixative solutions containseveral different fixing agents in combination.

Addition

and

cross-link

formation

The

non-coagulant

fixing agents chemically react with proteins and other cell and

tissue

components,

becoming

bound

to

them

by

addition and forming inter-molecular and intra-molecular cross-links. Because these agents are reactive compounds they bind to a variety of chemical groups in tissues, oftenaffecting the charge at the site of attachment.

This

can

have

an

effect

on

the

subsequent

staining

characteristics of a particular protein as well as altering itsmolecular

conformation

and

thus

its

solubility.

For

example, tissue fixed with formaldehyde stains poorly witheosin because formaldehyde reacts extensively with aminogroups to form methylene bridges and thus these groups areno longer available to bind negatively charged dye moleculessuch as those of eosin

Eosin

Time interval •

The optimal time for fixation will vary between fixatives. Forfixation to occur the fixative has to penetrate, by diffusion, tothe centre of the specimen and then sufficient time has to beallowed for the reactions of fixation to occur.

Both

diffusion

time

and

reaction

time

depend

on

the

particular reagent used and the optimum time will vary fromfixative to fixative. In busy diagnostic laboratories there isconsiderable pressure to reduce turnaround time and this canresult in incompletely-fixed tissues being processed.

This can lead to poor quality sections showing tissue distortionand poor quality staining because poorly fixed tissue does notprocess well.

However, the longer you wait, the more cellular organelles willbe lost and the more nuclear shrinkage.

Penetration Penetration of tissues depends upon the diffusability of each individualfixative, which is a constant. Formalin and alcohol penetrate the best, andglutaraldehyde the worst. Penetration into a thin section will occur morerapidly than for a thick section. Volume The volume of fixative is important. It should be a 1:10 ratio of fixative totissue. Concentration Concentration of fixative should be adjusted down to the lowest levelpossible, to reduce expenses. Formalin is best at 10%; glutaraldehyde isgenerally made up at 0.25% to 4%. Too high concentration may adverselyaffect the tissues and produce artifact. Specimen dimensions The preceding approximations emphasise the importance of specimendimensions when fixing tissue. A specimen should not be more than 4 mmthick. Ideally a 3 mm thick slice should provide excellent fixation andprocessing. It is useful to remember that the specimen cavity in a standardprocessing cassette is 5 mm deep.

PRACTICAL PROCEDURES TO OPTIMISE FIXATION

Essential 1: Fresh tissue

Essential 2:Proper

penetration of fixative

Essential 3: Right

choice of a correctly

formulated fixative

Fix quickly

.

degeneration

commences

as

soon

as

cells

are

deprived

of

a

blood

supply.

Cavities should be

opened.

Fixatives should be

carefully made up

from reagents of

suitable quality

If fixation is not

immediately possible

refrigerate, do not

freeze.

damage due to the

formation of ice crystals

Perfusion of some

specimens is

advantageous.

Specimens received in

fixative should be

checked.

Infection.

incompletely-fixed tissue

as potentially infectious

An adequate volume is

vital

Fixatives should be

used once only.

Dryness

cause permanent damage

and may mask

pathological change.

Some agitation is

useful.

Avoid metal lids.

4-TISSUE PROCESSING^

Post-fixation (Washing)

Fixatives

have

the

potential

to

further

react

with

any

staining

procedure,

which

may

be

used

later

in

the

process.

Thus

any

remaining fixative must be washed out in a process named post-fixation which differ according to the type of the fixative:

• Bouin-fixed tissues should be washed several times in 70% ethylalcohol till the removal of the yellow color of the picric acid.