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Microtechniques (microscopic preparations)

Purpose to study Micro-techniques

A) In general :

Students can prepare permanent slides for different body organs.

B) Specifically student can do:

1-permanent stained tissue slide and body fluid smears .

2- Preparing all needed chemical solution .

3- Fix and preserve tissue specimen.

 This technique is closely related to many sciences such as

pathology, histology, and cytology and other sciences served by
microscopic preparations, had to be defined Some of these sciences:

Cytology: The study of cells; their origin, structure and function.

Histology: The microscopic study of normal tissues of body .

Pathology: The scientific study of effecting of disease an

abnormal variation in the structure or Function or any part of the
body .

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 Cytopathology: scientific study of an abnormal variation in the
structure or function of cell.

Histopathology: The microscopic study of the tissue effecting by

diseases.

Microscopic slide: The most effective mean of study normal and

diseased tissue of the body microscopically is by the examination
of this section and stained to demons certain and to get good
results.

Microtechnique: (methods) used in preparing microscopically

section from organ & blood smear, different tissues.

Specimen: A tissue using in preparation of microscopically slide.

References of specimens :
1. Biopsy: cutting of a small piece of living tissue for microscopic
examination.
2. Autopsy: examination of the body of an tissue removed from the
body after death .
3. Laboratory animals.

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Microscopic preparations are divided into two methods:
1. Non-Sectioning method: Sections cannot be made in it like blood
tissue.
2. Sectioning method: pertaining to light microscope and electron
microscope.

Methods are used in the preparation of microscopic slide :

1. The Paraffin method (This is the most popular method).
2. The Freezing method(It is the fastest(.
3. The Celloidin method(It is the most accurate).
4. Electron microscope method.

The Paraffin Technique

In this technique, molten and solid paraffin wax is used.

Advantages of Paraffin wax method:

1- Very thin sections may be obtained.
2- The processes is very quick.
3- It is easy to get serial sections.
4- The tissue blocks can be easily stored

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 Disadvantages of paraffin wax method:
1- Overheated paraffin renders the specimen brittle making
sectioning difficult.
2- Long treatment in paraffin causes shrinkage and hardensening
of the tissue.
3- Tissue that are difficult to infiltrate (bone, teeth, eye, brain)
need long immersion for support.
4- Paraffin process removes fat (the dehydrants and clearing
agents) used in this process are fat solvents.

The steps involved are used to make tissue sections on glass slides:

1. Fixation
2. Washing
3. Dehydration
4. Clearing
5. Infiltration
6. Embedding(Blocking)
7. Trimming
8. Sectioning (Microtomes)
9. Staining
10. Mounting & Covering

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 Fixation
The first step is to prepare the tissues to treat the sample with fixation
materials to preserve the tissue and its contents as it in the body of the
living organism.

 Aim of fixation :-
1. Confers chemical stability on the tissue
2. Hardens the tissue (helps further handling)
3. protect the cells from distortion and shrinkage when they are
subjected to alcohols and hot paraffin.
4. prevent changes after death (post mortem changes) Such as:-
a. Autolysis (by enzymes)
b. Putrefaction (by bacteria)
c. Disintegration

 Factors affect fixation: According to previous factors we can

select the concentration of fixative and fixation time.
1. PH.
2. Temperature.
3. Penetration of fixative.
4. Volume of tissue .

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 Types of fixatives :-
A. According to the coagulation of protein

1- Coagulant fixatives:- These fixatives convert homogenous spongy

shape of protein to insure the penetration of the wax into the tissue
(like mercuric chloride, ethyl alcohol).
2- Non coagulant fixatives:- These fixatives coagulated the protein
into normal shape (like formaldehyde – acetic acid).

B. According to the chemical component

1- Simple fixative:- Contain one chemical substance like:-
(formaldehyde, mercuric chloride, acetic acid, picric acid, ethyl
alcohol)

2- Compound fixative:- Contain more than one chemical substance

like
(Zinger, HelleyFluid, Bouin, Carnoy)

Washing
It means the removal of the excess of uncombined fixatives and
precipitate of some fixatives.
the type of washing solution depending on the type of fixative:

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No Type of fixative Type of washing solution

1 10% formalin running tap water

2 Zenker running tap water and 70%

alcohol

3 Helly running tap water and 70%

alcohol

4 Bouin 70% alcohol

5 Carnoy 100% alcohol

Dehydration
Dehydration: is removing of excess water from the tissue.
Tissues are dehydrated by using increasing strength of alcohol; e.g.
50%, 70%, 90% and 100%. The duration for which tissues are kept in
each strength of alcohol depends upon

1.the size of tissue,

2. fixative used

3. type of tissue; e.g. after fixation in aqueous fixative delicate tissue

need

to be dehydrated slowly starting in 50% ethyl alcohol directly whereas

most tissue specimens may be put into 70% alcohol. Delicate tissue

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will get high degree of shrinkage by two great concentration of
alcohol.

• The volume of alcohol should be 50-100 times that of tissue

1.Tissues contain much "free" water which does not mix well with the
paraffin used later in the procedure. Therefore, water in the tissues
must be removed by submerging the tissue in alcohol, a process
known as alcohol dehydration.

2.If this is done too rapidly, the large outflow of water can damage the
morphology of the cells and tissues.

3.In this step, tissue is placed into a series of gradually increasing

concentrations of alcohol, usually ethyl alcohol (30, 50, 70 ,80, 95,
and 100%), for specific periods of time.

Dehydrating agents:-
1. Alcohol;.

2. Acetone: (dehydrate quickly).

3. Diaxone

4. Tetranydrofuran:- like dioxane.

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 Clearing
CLEARING:(DE- ALCOHOLIZATION) It is the removal of
dehydrating agent from the specimen and filled with fluid can mixed
with paraffin wax and makes the specimen translucent allow the
microscope light to penetrate to insure a good diagnosis or study the
specimen.

Clearing agents:

1. Xylol (xylene):- colorless with special order.

2. Chloroform:- slow in clearing than xylol for this must be put

the specimen long time in it.

3. Benzene:- more speed in clearing, but effect on the bone marrow

when treat with it for long period.

4. Toluene:- very expensive.

5. Dioxane:- used in hydration and clearing.

6. Cleve oil.

7. Cedar wood oil:- these two clearing agent are difficult to remove

from the specimen after used for thin must be used three

steps in paraffin fluid when infiltrate.

8. isopropanol:- used in dehydration and clearing.

9. Tertiary butyl.
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 Infiltration
After tissue have been thoroughly cleared with clearing agent(xylene)
for example it is necessary to infiltrate the tissue with supporting
medium to hold the cell and inter cellular structure in proper relation
to each other, for this purpose embedding media is used paraffin wax.

The aim of this step is for supporting the tissue against harmful of the
embedding media and facilitate the cutting.

Infiltration or embedding media:-

Paraffin wax – Collidin – Gelatin – Plastic.

Type of paraffin wax:-

There are three types of paraffin wax according to melting point:

1. Soft P. W. The melting point between 48 – 50 c°.

2. Medium P.W. The melting point between 50–56 c°.

3. Hard P. W. The melting point between 56 – 60 c°.

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 Embedding (Blocking – Casting)

Following complete infiltration the specimens are cast in moulds

(block) for easy in cutting by microtome.

Type of embedding:-
1. Simple embedding and infiltration with same media.
2. Compound embedding infiltration with certain media. Like
celloidin and the embedding by other media. Like wax.
3. Embedding with paraffin wax famous.

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 Trimming
It is the removing of the excess wax from the block by shaving the
edge of the block from an surface for making the paraffin ribbon.

Sectioning(Cutting)
Cutting the block:-
This process is used for cutting very thin sections of tissues into micro
section with thickness between (1 – 50( μ using a tool called
microtome.

Microtome:- is a machine specifically designed to cut very thin

sections of tissue.

Types of microtome:-
We can classified the microtome according to the types of embedding
Medium to:-
1. Rotary microtome: for cutting paraffin embedded sections with(3-8
Microns).
2. Sliding microtome: for cutting celloidin embedded section with (8-
15 Microns ).
3. Freezing microtome: for cutting un embedded tissue that have been
frozen with (CO2 gas), Freon or other refrigents (10-15 microns).

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 4. The cold microtome (Cryostat): is a rotary microtome mounted
within a refrigerated Chamber for cutting uniformly thin sections of
un embedded fresh or fixed tissue.
5. Ultra-microtome: for cutting tissue preparation by electron
microscope method with (600-900 A).

MICROTOME KNIVES:

Types of knifes:- We can classified them according to their

cross-section as follows
1. Plano- concave knife: used for (P.W. or Celloidin ) medium.
2. Wedge- shape knife: used for (P.W. , Celloidin and frozen
sections.
3. Bi- concave knife: used for (P.W.) only.
4. Knife with diamond or glass cutting in special angle: used for
(Ultra) sections.

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