FIXATION

Question 1

• The first & most critical step; Basis of histological techniques.

Answer 1

FIXATION

Question 2

• It is done after the process of gross examination.

Answer 2

FIXATION

Question 3

It is the method of preserving cells & tissues in life-like states as possible (it is the first crucial stage in tissue processing).

Answer 3

Fixation

Question 4

• It is the process by which the cell & tissue constituents are preserved with the least alteration from the living state with the use of fixatives.

Answer 4

Fixation

Question 5

prevents the breaking down of cellular elements via autolysis by inactivating lysosomal enzymes, making the tissue components insoluble.

Answer 5

Fixation

Question 6

must render the specimen insensitive to subsequent treatment as may be necessary to permit microscopic examination.

Answer 6

• Fixatives

Question 7

Factors Affecting Fixation

Answer 7

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Penetration rate
Volume of the fixaitve
Osmolality
Temperature
Time
Size of the specimen

Question 8

Types of Fixatives

Answer 8

Aldehyde
Alcohols
Acetic acid
Acetone

Chromates
Osmium tetroxide
Mercuric chloride
Picrates

Question 9

pH

The process should be kept in the physiological range, between the

Answer 9

pH: 6 to 8 (neutral pH)

Question 10

pH

The pH for the ultrastructure preservation should be buffered between

Answer 10

7.2 to 7.4.

Question 11

is the most commonly used fixative in the histopathology laboratory.

Answer 11

formalin

Question 12

Unbuffered formalin will slowly oxidize to formic acid resulting in a fall in pH.

Under these conditions, the formic acid will react with hemoglobin, forming:

Answer 12

Acid formaldehyde hematin

Question 13

The osmotic effects exerted by the fixative are more important at the ultrastructural level than at the level of the light microscope because it is the phospholipid membranes that are easily damaged by excessively________

Answer 13

hypotonic or hypertonic solutions.

Question 14

osmolality of the vehicle (____)

Answer 14

buffer

Question 15

It is the ______ of the vehicle (buffer) that is most important & in some formulations, this is adjusted to resemble that of tissue fluid (e.g., formalin is isotonic saline).

Answer 15

osmolality

Question 16

Before fixation occurs, cells can certainly be damaged by non-isotonic fluids such as water & if specimens cannot be immediately fixed, they can be kept moist with gauze soaked in __________ for a short time.

Answer 16

isotonic saline

Question 17

Size

A specimen should not be more than_____ thick.

Answer 17

4mm

Question 18

Size

Ideally, a_____ thick slice should provide excellent fixation & processing.

Answer 18

3mm

Question 19

Size

It is useful to remember that the specimen cavity in a standard processing cassette is____ deep.

Answer 19

5mm

Question 20

Volume of fixative

A fixative-to-tissue ratio of _____is considered the lowest acceptable ratio.

Answer 20

20:1

Question 21

Temperature

Increasing the temperature of_____ will increase the________ of the fixative into the tissue & speed up the rate of______between the fixative & tissue elements.

Answer 21

fixation

rate of diffusion

chemical reaction

Question 22

It can also potentially increase the rate of tissue degeneration in unfixed areas of the specimen.

Answer 22

Temperature

Question 23

Fixation is routinely carried out at_____ temperature (recommended temperature).

Answer 23

room

Question 24

may involve the use of higher temperatures (up to 65°C), but for relatively short periods.

Answer 24

Microwave fixation

Question 25

Microwave fixation may involve the use of higher temperatures (up to_____), but for relatively short periods.

Answer 25

65°C

Question 26

T or F The optimal time for fixation will vary between fixatives.

Answer 26

True

Question 27

For fixation to occur, the fixative has to______, by diffusion, to the center of the specimen and then sufficient time has to be allowed for the reactions of fixation to occur.

Answer 27

penetrate

Question 28

Formalin fixation usually takes up about ____

Answer 28

24 hours

Question 29

_______of a fixing agent depends on its diffusion characteristics & varies from agent to agent.

Answer 29

penetration rate

Question 30

penetration rate of formalin is_____

Answer 30

1 mm/hr.

Question 31

act by creating covalent chemical bonds between proteins in tissues.

Answer 31

Aldehyde fixatives

Question 32

This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue.

Answer 32

Aldehyde

Question 33

Different types of aldehyde include:

Answer 33

1. Formaldehyde 2. Glutaraldehyde

Question 34

Formaldehyde The only gaseous aldehyde & is dissolved in water to saturation at

Answer 34

37% to 40% w/v.

Question 35

This solution is generally referred to as "formalin" or "concentrated formaldehyde solution".

Answer 35

37% to 40% w/v.

Question 36

For fixation, 1 part formalin is usually diluted with___ parts of water or buffer.

Answer 36

9

Question 37

, a highly polymerized form of formaldehyde may be deposited as a white precipitate in concentrated formaldehyde solutions.

Answer 37

Paraformaldehyde

Question 38

FIXATION: PROCEDURE

Answer 38

Examine the tissue grossly. Cut a representative specimen into 2cm2, but not more than 4mm in thickness. Place the specimen inside the cassette. You may also opt to label the plastic cassette with a pencil instead of attaching the piece of paper. Fill a 500mL beaker w/ 10% formalin up to the 400mL mark. Submerge the tissue-filled cassette into the solution. Cover the beaker with foil. Label it with your group number & section. Send it to the laboratory for storage until the next laboratory session.

Question 39

Unbuffered formalin will slowly oxidize to———— resulting in a fall in pH. Under these conditions the formic acid will react with hemoglobin forming————-, a brown-black granular artefact pigment which is deposited in blood-rich tissues.

Answer 39

formic acid acid formaldehyde hematin

Question 40

formaldehyde reacts most effectively at about a neutral pH, 10% formalin solutions are usually buffered to

Answer 40

pH 6.8 – 7.2.

Question 41

Studies have shown that ——— causes nasopharangeal cancer, sinonasal cancer and myeloid leukaemia. For these reasons in most countries there are strict guidelines innplace to limit the exposure of workers to ——— in the workplace

Answer 41

formaldehyde

Question 42

is a larger molecule, and so its rate of diffusion across membranes is slower. It is recommended that tissue be less than 1mm in thickness in at least one dimension.

Answer 42

glutaraldehyde

Question 43

One of the advantages of———- fixation is that it may offer a more rigid or tightly linked fixed product. It causes rapid and irreversible changes, fixes quickly, is well suited for electron microscopy, fixes well at——, and gives best overall cytoplasmic and nuclear detail. However it is not ideal for———— staining.

Answer 43

glutaraldehyde 4ºC immunohistochemistry

Question 44

For electron microscopy glutaraldehyde primary fixation is commonly followed by secondary fixation in——-

Answer 44

osmium tetroxide

Question 45

Some fixation protocols call for a combination of———-&———- so that their respective strengths complement one another. These crosslinking fixatives–especially formaldehyde–tend to preserve the secondary structure of proteins and may protect significant amounts of tertiary structure as well.

Answer 45

formaldehyde and glutaraldehyde

Question 46

Precipitating (or denaturing) fixatives act by reducing the solubility of protein molecules and (often) by disrupting the hydrophobic interactions that give many proteins their tertiary structure. The precipitation and aggregation of proteins is a very different process from the crosslinking that occurs with the aldehyde fixatives.

Answer 46

Alcohol

Question 47

The most common precipitating fixatives are——-&——-

Answer 47

ethanol and methanol

Question 48

They are commonly used to fix frozen sections and smears. Fixation commences at a concentration of 50 – 60% for ethanol and >80% for methanol.

Question 49

They are commonly used to fix frozen sections and smears. Fixation commences at a concentration of 50 – 60% for ethanol and >80% for methanol.

Question 50

is sometimes used to preserve glycogen but will cause distortion of nuclear and cytoplasmic detail.

Answer 50

Ethanol

Question 51

———% is used as a fixative for cytology smears.

Answer 51

95% ethanol

Question 52

———% is used as a fixative for cytology smears.

Answer 52

95% ethanol

Question 53

is commonly used as a fixative for blood films. Both alcohols are usually combined with other reagents when used as fixatives for tissue specimens.

Answer 53

Methanol

Question 54

is coagulant in action with nucleic acids but generally does not fix proteins. It is incorporated in compound fixatives to help prevent the loss of nucleic acids and, because it swells collagen, to counter the shrinkage caused by other ingredients such as ethanol.

Answer 54

Acetic acid

Question 55

penetrates very rapidly but fixatives that contain it will lyse red blood cells.

Answer 55

Acetic acid

Question 56

It is a highly toxic, volatile, crystalline solid which is supplied in sealed ampoules. Because of its volatility it must be handled with great care under a fume hood because it will readily fix the conjunctiva of the eye and the nasal mucosa.

Answer 56

OSMIUM TETROXIDE

Question 57

The most important fixation reactions of_______ are those involving unsaturated bonds of lipids and phospholipids as it is one of the few fixatives that stabilises lipids

Answer 57

osmium tetroxide

Question 58

During fixation _______is reduced to lower oxides which are black and insoluble and these are deposited in tissues, particularly on membranes.

Answer 58

osmium tetroxide

Question 59

_______ is a heavy metal it scatters electrons and thus adds electron density to the electron microscope image. It can also be used as an en bloc stain for demonstrating_____ (particularly myelinated nerve fibres) at the light microscope level.

Answer 59

osmium lipids

Question 60

Generally it is used at about 1% w/v as a specialised secondary fixative for electron microscopy and for teased nerve fibre preparations.

Answer 60

Osmium

Question 61

It is a powerful protein coagulant which leaves tissue in a state which produces strong staining with acid dyes.

Answer 61

Mercuric chloride

Question 62

It reacts with phosphate residues of nucleic acids and effectively fixes nucleoproteins. It is for this reason that it is the major component in formulated fixatives such as B-5 and Helly’s, fixatives recommended when high quality nuclear preservation is required

Answer 62

Mercuric chloride

Question 63

Apart from the corrosive nature of________, it is highly toxic, can be absorbed through the skin and is a cumulative poison

Answer 63

mercuric chloride, mercury

Question 64

During fixation with fixatives containing_______ a crystalline or amorphous greenish-brown artefact pigment of _____ is randomly deposited in tissues.

Answer 64

mercuric chloride; mercuric chloride

Question 65

Treatment of specimens with______ during processing or sections prior to staining, will produce mercuric iodide which can be washed out of the tissues. A subsequent treatment with_______ removes residual iodine.

Answer 65

Lugol’s iodine sodium thiosulphate

Question 66

_______-based fixatives tend to penetrate poorly and if fixation is prolonged tissues become very hard and are prone to shrinkage during processing.

Answer 66

Mercuric chloride

Question 67

is a bright yellow crystalline substance that must be stored wet with water to avoid the risk of explosion by percussion or heating of the dry substance It should be kept in a tightly sealed container and regularly dampened with distilled water

Answer 67

Picric acid

Question 68

For fixation, it is always used in combination with other agents. It imparts a______ color to tissues during fixation and because of its acidic nature residual picric acid should be washed from tissues with______ before processing.

Answer 68

yellow 70% ethanol

Question 69

Picric acid appears not to fix____ or most_______ but is recommended as a component in fixatives used to preserve______.

Answer 69

lipids carbohydrates glycogen

Question 70

Picric acid can hydrolyze nucleic acids so it should be avoided if ________are to be demonstrated. It will dissolve small deposits of____ in specimens and a considerable amount of shrinkage occurs during the processing of tissues fixed in picric acid containing reagents.

Answer 70

DNA or RNA calcium

Question 71

has a similar action to alcohol and has been used as a fixative and dehydrant for tissue processing, particularly rapid hand-processing of small specimens

Answer 71

Acetone

Question 72

It is widely recommended for fixation as part of the histochemical demonstration of enzymes where it is generally used cold (4°C).

Answer 72

Acetone

Question 73

It is an effective lipid solvent with a rapid action which can make tissues very brittle. Because it is highly volatile and flammable it is generally not used on automatic tissue processors

Answer 73

Acetone