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MICROTECHNIQUE

MICROTECHNIQUE

Soran UniversityFaculty of Science and Engineering

Biology departmentMicrotechnique in medical and biology

MICROTECHNIQUE

(In biology), the aggregate of the methods and procedures used

to study the structure, vital activity, development, chemical

composition, and physical properties of cells, tissues, and

organs by means of optical and electron microscopes.

Generally the microtechnique in medical is the science that

studies the way to deal with the human specimen and to prepare

it to be studied by the pathologist under the light microscope.

Microtechnique in Medical

The microtechnique is divided to two main ways:

-1- Sectioning method-2- Non sectioning method

•The sectioning method is the way we deal with the solid sample

(tissue) which needs to be sectioned by the microtome to obtain very

thin sections which can be studied under light microscope.

•The non sectioning method is the way to deal with the human body

fluid, (like: pleural effusion, ascetic fluid, C.S.F,,,, et) and these

specimens don’t need to be sectioned.

Sectioning Method •Obtaining the sample.

•Fixation.

•Dehydration.

•Clearing.

•Infiltration

•Embedding.

•Sectioning.

•Staining.

•Mounting.

Obtaining the sample

We can take the samples from living patient (biopsy), and from dead body (autopsy).

The biopsy: is a tissue (part of organ or an entire organ) removed by the surgeon in the operating room under general or local anesthesia.

This tissue is suspicious to have abnormality (infection, congenital abnormalities or a tumoral proliferation).

Obtaining the sample

The biopsy is two types:

•Excisional biopsy: remove all the suspicious area.

•Incisional biopsy: take small piece of the suspicious lesion.

And these biopsies send to be studied under the light microscope by the pathologist.

Obtaining the sample

The autopsy: is known as post-mortem examination in

which the physician take a samples from dead human

body (from the organs which suspicious to cause the

death) and send to the pathology department to

examined and to determine the cause and the manner

to death.

Obtaining the sample

After we receive the specimen:

If the specimen is small specimen, entire must submitted.

If the specimen is large we must take good sections from this biopsy (good section: thin, adequate, consist of all the histological changes and all tissue types)

So: We must have anatomy knowledge to select the good location of our sample.

And also we must write any explain about the shape and size and any abnormalities in our specimen.

And it is better to take a photo to it.

TISSUE FIXATION

• Fixation is a complex series of chemical events that differ for the different groups of substance found in tissues.

•The aim of fixation:

1- To prevent autolysis and bacterial attack. 2- To fix the tissues so they will not change their volume and shape during processing. 3- To prepare tissue and leave it in a condition which allow clear staining of sections. 4- To leave tissue as close as their living state.

•Fixation is coming by reaction between the fixative and protein which form a gel, so keeping every thing as their in vivo relation to each other.

TISSUE FIXATONType of fixation

There are generally three types of fixation process:Heat fixation: After a smear has dried at room temperature, the slide passed through the flame of a Bunsen burner several times to heat-kill and adhere the organism to the slide. Routinely used with bacteria and archaea. Heat fixation generally preserves overall morphology but not internal structures. Heat denatures the enzyme and prevents autolysis.

Perfusion: Fixation via blood flow. The fixative is injected into the heart with the injection volume matching cardiac output. The fixative spreads through the entire body, and the tissue doesn't die until it is fixed. This has the advantage of preserving perfect morphology, but the disadvantages that the subject dies and the cost is high

Immersion: The sample of tissue is immersed in fixative of volume at a minimum of 20 times greater than the volume of the tissue to be fixed. The fixative must diffuse through the tissue to fix, so tissue size and density, as well as type of fixative must be considered.

TISSUE FIXATION

PH.Should be kept in the physiological range, between pH 4-9. The pH for the ultra structure preservation should be buffered between 7.2 to 7.4

Temperature.Increasing the temperature increases speed of fixation. However, care is required to avoid cooking the specimen. Fixation is routinely carried out at room temperature.

Penetration of fixative.As a general rule, 1hr per 1mm of tissue must penetrate.

Volume of tissue.At least 15-20 times greater than tissue volume.

The Factors that affect fixation:

TISSUE FIXATION

Characteristics of a good fixative

1-It must kill the cell quickly without shrinkage or swelling.

2-It must penetrate the tissue rapidly.

3-It must inhibit bacterial decay and autolysis.

4-Harden the tissue and render it insensitive to subsequent treatment as staining.

5-It should allow tissue to be stored for long time.

6-It should be simple to prepare and economical is use.

TISSUE FIXATION

Types of fixative:

Acetic acid,

Formaldehyde,

Ethanol,

Glutaraldehyde,

Methanol and Picric acid.

TISSUE FIXATION

Formalin: Of the many fixatives that have been proposed, 10% buffered formalin remains the

best compromise under most circumstances. It is inexpensive, the tissue can

remain in it for prolonged periods without deterioration, and it is compatible with

most special stains, including Immunohistochemical techniques, as long as the

tissue is placed in fixative shortly (<30 min) after surgical removal, and over

fixation (>24–48 hours) is avoided.

Pure’ formalin is a concentrated (40%) solution of the gas formaldehyde in water. Thus a 10% formalin solution represents a 4% solution of the gas, which is 1.3 molar.

TISSUE FIXATION

Other fixative agents

Zenker fluid (which incorporates mercuric chloride) is an excellent fixative, one of the best that has ever been devised for light microscopic work, but it is expensive, requires careful disposal of the mercury, and necessitates meticulous attention to fixation times and washing procedures to remove the precipitates of mercury.

Carnoy fixative is a mixture of ethanol, chloroform, and glacial acetic acid. Thus at the same time that it fixes the tissues, it dissolves most of the fat. This property has been found useful for the identification of lymph nodes in radical resection specimens.

TISSUE FIXATION

Bouin fixative (which contains picric acid) has been especially recommended

for testicular biopsies, but Zenker fluid results in almost identical preparations.

Bouin, Zenker, and B-5 are excellent fixatives for routine work and for most

immunohistochemical stains, but the preservation of nucleic acids is very

poor.

WASHING

Following fixation, the tissues should be washed from 15 to 30

minutes. The fixed tissues are washed in running tap water to

remove the fixative from them.

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 usual way this is done is with paraffin. Tissues embedded in

paraffin, which is similar in density to tissue, can be sectioned at

anywhere from 3 to 10 microns, usually 5-7 routinely. The technique of

getting fixed tissue into paraffin is called tissue processing.

TISSUE PROCESSING

Stages of processing:

1- Dehydration.

2- Clearing.

3- Embedding.

DEHYDRATION

So in dehydration we remove fixative and water from the tissue and replace them with dehydrating fluid. There are a variety of compounds many of which are alcohols. Several are hydrophilic so attract water from tissue.

Wet fixed tissues (in aqueous solutions) cannot be directly infiltrated

with paraffin. Because melted paraffin wax is hydrophobic (immiscible with

water),

First, the water from the tissues must be removed by dehydration.

DEHYDRATION

To minimize 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 stage.

DEHYDRATION

Types of dehydrating agents:

Ethanol, Methanol, Acetone.

•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 .

CLEARING

•Replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium.

The term “clearing” was chosen because many (but not all) clearing

agents impart an optical clarity or transparency to the tissue due to

their relatively high refractive index. Another important role of the

clearing agent is to remove a substantial amount of fat from the tissue

which otherwise presents a barrier to wax infiltration.

CLEARING

- Safety factors.

- Cost and convenience.

- Speedy removal of dehydrating agent.

- Ease of removal by molten paraffin wax.

- Minimal tissue damage.

CLEARING•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 pressure.

CLEARING•Some clearing agents:

- Xylene. - Toluene. - Chloroform. - Benzene. - Petrol.

Xylene

A rapid clearing agent suitable for urgent biopsies . It is cheap and

highly flammable . Tissues are rendered transparent by xylene and it

volatilizes readily in the paraffin oven . Biopsies , and tissue blocks not

exceeding 3mm in thickness , are cleared in 15-30 min but some material

, notably brain and blood- containing tissues , tends to become brittle if

immersion is prolonged.

Benzene

A popular routine clearing agent until recently when its highlycarcinogenic properties were recognized. Its use for clearing purposes is therefore strongly discouraged .

Toluene

This appears to have superseded benzene as an ante-medium forroutine work because of its lower toxicity . Like xylene it is highlyflammable , has similar clearing properties , but without the same brittle effect on tissues . It is somewhat more expensive than xylene . Clearing time is from 15 to 180min , depending on tissue type and thickness .

CLEARING

INFILTRATION WITH PARAFFIN

The final stage in tissue preparation is to prepare specimens which allow the cutting of sections thin enough for microscopy.

In the process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning.

Tissue infiltration is always done with liquid media. In the case of paraffin, tissue blocks are treated with hot liquefied which becomes solid when cooled down to room temperature. Paraffin with different hardness and melting points are available; the higher the melting point, the harder the wax. In general practice, paraffin with a melting point of 56°C are used.

INFILTRATION WITH PARAFFIN

In this stage we replace xylene with paraffin so first we must immerse the sample in melted paraffin for 30 minutes to 1 hour. This step should be repeated.

EMBEDDING

The choice of embedding substance depends mainly on the type of histological study to be performed. Paraffin wax is the usual embedding medium for histopathology and many other light microscopical purposes.

Paraffin wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. It is about two thirds the densities and slightly more elastic than dried protein. Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C.

EMBEDDING

•The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax:

- improve ribboning. - increase hardness. - decrease melting point - improve adhesion between specimen and wax

Precaution while embedding in wax

•The wax is clear of clearing agent.•No dust particles must be present.•Immediately after tissue embedding, the wax must be rapidly cooled to reduce the wax crystal size.

EMBEDDING•There are four main mould systems and associated embedding

protocols presently in use :

1- Traditional methods using paper boats

2- Leuckart or Dimmock embedding irons or metal containers

3- The Peel-a-way system using disposable plastic moulds and

4- Systems using embedding rings or cassette-bases which become an

integral part of the block and serve as the block holder in the microtome.

EMBEDDING

Today we will use Dimmock or Leuckart embedding mould to make block. 1. Place tissue in mold.2. Fill mold with melted paraffin.3.Allow to cool.

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)

Histological sections refers to be thin slices of tissue which applied to the microscope slide, usually around 3 -5 -6 micrometers thick, to be viewed under light microscope after staining.

We can take sections in tissue through different planes:1 – Transverse (cross): sectioning is vertical to the longitudinal axis.2 – Sagittal (longitudinal): sectioning is parallel to the longitudinal axis.3 – Frontal : sectioning is parallel to longitudinal axis and including right and left site of the sample.

  MICROTOMING AND MICROSECTIONING

The embedding position of the sample of tissue and the sectioning direction is responsible of these various sectioning planes.

  MICROTOMING AND MICROSECTIONING

Microtome sectioning

A microtome is a mechanical instrument used to cut biological specimens into thin transparent sections for light microscope examination. Microtome use steel, glass or diamond blades (knifes) depending on the specimen being sliced and to desired thickness of the sections being cut. Steel blades are used to prepare sections of animal or plant tissue for light microscope histology. Glass knifes used to slice sections for light microscope or very thin sections for electron microscope. Diamond knife are used to slice hard materials such a bone, teeth, and plant matter for both light and electron microscope.

  MICROTOMING AND MICROSECTIONING

Types of microtome:1-rotary microtome:

One in which wheel action is translated into back-and-forth movement of the specimen being sectioned. It has a stationary knife holder and movable block holder clump with accurate working mechanism. The adjustment which controls the cutting thickness is usually located on the front panel of the microtome and is graduated from 1 to 25 or 35 micrometers in step of one micrometer. These machines are ideal for cutting hard tissue and soft tissue. The produced sections are appeared as almost perfect flat ribbon easy to handle with. Before cutting the paraffin block, it must have a hard texture, so it must be put into the ice box for hours.

  MICROTOMING AND MICROSECTIONING

2-Freezing microtome: (cryostat)A microtome used to cut frozen tissue. These microtomes freeze the specimens to harden it, so it can be slices without ripping and destroying it. Unlike rotary microtome: freezing microtome has a movable knife holder and stationary block holder.

3-Ultramicrotome:Ultramicrotome is similar to rotary microtome but the knife are used here is either glass (which could be made in the lab by suing knife maker) or a diamond. This microtome used to sectioning the samples embedded in plastic block.

1)Microtome base plate or stage: A platform which has rails that secure the

knife holder base.

2)Knife holder base: A part that anchors the knife holder to the microtome

stage. The knife holder base can be moved toward or away from the block, but

MUST be stationary and locked during microtomy.

3)Knife holder: This part is comprised of several components including the

blade clamp that holds the blade, the knife tilt for adjusting the knife angle, and

the face plate that guides that ribbons away from the blade and towards the

operator.

  MICROTOMING AND MICROSECTIONING

The parts of rotary microtome

  MICROTOMING AND MICROSECTIONING

4)Cassette clamp or block holder: Holds the paraffin block in

place. Typically, the block moves up and down with each

revolution while the blade is stationary. The block holder may have

knobs that allow the user to manipulate the block face in various

directions to bring the tissue in alignment with the blade.

5)Micron adjustment: Micron settings for section thickness can range from 1 to 60 microns on most microtomes.

  MICROTOMING AND MICROSECTIONING

1.    Knife regulating rod for inclining angle 2.    Sliding knife holder 3.    Holder regulating knob 4.    Holder locking rod 5.    Knife fixing knob 6.    Paraffin block transversal regulating knob 7.    Paraffin block fixed locking rod 8.    Paraffin block fixing screw 9.    Paraffin block vertical regulating knob 10.  Rotary hand wheel 11.  Sectioning thickness regulating knob 12.  Hand wheel locking rod

  MICROTOMING AND MICROSECTIONING

40o C water bath

Flattens paraffin section (put on water to

stretch the section)

Carefully pick up on a clean slide

- Cut the tissue with microtome

-Put the slides into a rack

-Dry slides at 65 for 30 minutes -2 hours℃

H&E STAINING

3)Deparaffinize in Xylene I and II and III (5 minutes)4)Rehydrate

EtOH 100% (3 minutes)EtOH 100% (3 minutes)EtOH 95% (3 minutes)EtOH 95% (3 minutes)EtOH 70% (3 minutes)

6)Rinse in distilled water (5 minutes)7)Stain in Hematoxylin (6 minutes)

Hematoxylin and Eosin staining method

H&E STAINING

8)Rinse in running tap water (20 minutes)9)Decolorize in acid alcohol (1 second) Can go up to 3 seconds. 10)Rinse well in tap water (5 minutes)11)Immerse in Lithium Carbonate (3 Seconds)12)Rinse in tap water (5 minutes)13) Stain in Eosin (30 seconds)14)Dehydrate

EtOH 95 % (3 minutes)EtOH 95% (3 minutes)EtOH 100 % (3 minutes)EtOH 100 % (3 minutes)15)Clear in Xylene I and II (5 minutes)16)Drop 2-3 drops of mountant onto the slide and, then, put a cover glass onto the slide

The development of sciences lead us to more suitable

and rapid, correct method to do these various stages to

prepare tissue to the microscopic examination.

•Automatic tissues processor.

•Frozen section machine (Cryosection).

•Full automatic microtome.

• Auto- Stainer.

Non sectioning methods

•The non sectioning method is the way to deal with the

human body fluid, (like: pleural effusion, ascetic fluid,

C.S.F,,,, et) and these specimens don’t need to be

sectioned.

BUCCAL SMEAR

The cells which line the inside of your cheeks form a mucous

membrane and are classified as a stratified squamous epithelium

tissue. These flat, scale-like Buccal cells (pronounced "buckle") resist

friction and are shed constantly as the tissue is renewed. By gently

scraping the inside of your cheek, these cells can be harvested, and

when smeared and stained, may be used to illustrate a number of

important biological phenomena including cell and tissue structure,

oral bacterial flora and morphology, etc.

BUCCAL SMEAR

This tissue is non-keratinized and therefore the surface cells

are living and still possess their nuclei, in contrast with shed

epidermal cells.

EQUIPMENT

Soap and water

Microscope slides

Dropper bottle of dH2O

Dropper bottle of 0.3% methylene blue

Toothpicks or swap (optional)

Bunsen burner or alcohol lamp,

Paper towel (“non-linty”) or bibulous paper

SLIDE PREPARATION

1. Clean a microscope slide well with

soap & water, dry with a “non-linty”

paper towel.

2. Cleanse very thoroughly under the

nail of your index finger. (This step is

not necessary if you use swap or

toothpick)

3. Place a tiny drop of dH20 in the center

of the very clean slide.

HARVEST THE CELLS, PREPARE THE SMEAR

4. GENTLY scrap the inside of your cheek to pick up some of the shed

stratified squamous cells. Do NOT scrape chunks. A toothpick may be

used if you have no fingernails. Gentle scraping is the watchword, there

should be no discomfort...

5. Express the material from under your nail by pressing with your

thumb, and press into the drop of water, mix and spread the material

around to the size of a dime.

FIX AND STAIN THE SMEAR

FIX THE SMEAR:

6. Pass the slide briefly through the flame several times to warm and fix the

smear. Do NOT heat the slide above a temp which is comfortable. You are

"gluing" the smear to the slide. (you can also use 96% Methanol or

Ethanol for fixation)

STAIN THE SMEAR:

7. Place a drop of 0.3% methylene blue on the specimen. Let sit for 1

minute.

8. Rinse off the excess stain with tap water.

9. Blot dry with an non-linty paper towel or bibulous paper. Do not rub.

10. Flame again briefly to dry slide by warming.

EXAMINE UNDER THE MICROSCOPE:

11. Examine first with the 4x objective, scanning the entire field to find

a well-distributed region with individual cells (no big clumps). Then

view with the 10x and 40x objectives. Illustrate the 400x view noting 1)

the nucleus, 2) nucleolus, 3) cell boundary and 4) the variety of

bacteria colonizing the surface of the cells.

For Microbiology only: you will be instructed on oil immersion use,

then view and illustrate bacterial morphologies with the 100x oil

immersion objective (1000x view).

BUCCAL SMEAR

BUCCAL SMEAR

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Thank You!