FIXATION

Question 1

first and most critical step in histotechnology

Answer 1

Fixation

Question 2

a process that preserves tissues from decay, thereby preventing autolysis or putrefaction

Answer 2

Fixation

Question 3

results from tissue digestion by intracellular enzymes that are released when organelle membranes rupture

Answer 3

Autolysis

Question 4

brought about by microorganisms which may already be present in the specimen

Answer 4

Putrefaction or Bacterial decomposition

Question 4

Other term of putrefaction

Answer 4

Bacterial decomposition

Question 5

True or False
- strong salt cause the cell to swell

Answer 5

False
- water (hypotonic solution): cause the cell to swell

Question 6

True or False
- water cause the cell to shrink

Answer 6

False
- strong salt (hypertonic solution): cause the cell to shrink

Question 7

True or False
- in the case of smears, merely drying the preparation acts as a form of preservation

Answer 7

True

Question 8

List physical methods of fixation

Answer 8

1. Heating
2. Microwaving
3. Cryopreservation (freeze drying)

Question 8

rarely used on tissue specimens, its application being confined to smears of microorganisms

Answer 8

heat fixation

Question 9

can be regarded as a form of heat fixation, is now widely practiced in routine laboratories

Answer 9

microwave fixation

Question 10

A method of fixation where injecting the vascular system with fixative to small animals or some whole organs such as lung

Answer 10

Perfusion Fixation

Question 10

usually in the form of freeze drying, has some applications in histochemistry but is not usually applied to diagnostic tissue specimens

Answer 10

Cryopreservation

Question 10

List of chemical fixation

Answer 10

1. Immersing fixation
2. Perfusion Fixation
3. Vapor-Fix

Question 11

True or False
- alcoholic fixatives are examples of additive fixation

Answer 11

False
- non-additive fixation

Question 11

Mechanism of fixation where chemical constituent of the fixative is taken in and becomes part of the tissue by forming cross-links or molecular complexes and giving stability to the protein

Answer 11

Additive Fixation

Question 11

A mechanism of fixation where fixing agent is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attached to H-bonds of certain groups within the protein molecule

Answer 11

Non-additive Fixation

Question 12

_______________ is present in some formulations to counter the shrinkage caused by other agents such as ethanol

Answer 12

acetic acid

Question 13

True or False
- formalin, mercury, and osmium tetroxide are examples of non-additive fixation

Answer 13

False
- additive fixation

Question 14

How long should specimen be transferred to fixative quickly after surgery as deterioration will
commence with the loss of blood supply?

Answer 14

<1 hour

Question 14

Recommended volume of a fixative for penetration to occur in the most efficient manner?

Answer 14

20:1 or at least 10:1 fixative to specimen

Question 14

pH of fixation?

Answer 14

• fixation is best carried out close to neutral pH (6-8)

Question 15

most common error in histotechnology

Answer 15

insufficient ratio of tissue volume to fixative volume; 10-20 times the volume of tissue to be fixed

Question 15

True or False
- hypoxia of the tissue lowers the pH

Answer 15

True

Question 16

True or False - alkalinity favors formation of formalin-heme pigment that appears as black, polarizable deposits

Answer 16

False - acidity

Question 16

Temperature for electron microscopy and some histochemistry fixation?

Answer 16

0-4°C

Question 17

Common buffers use in fixation?

Answer 17

common buffers: phosphate, bicarbonate, cacodylate, and veronal

Question 17

Temperature for regular tissue processing fixation?

Answer 17

40°C

Question 17

Cells that are best fixed at room temperature even for electron microscopy

Answer 17

mast cells

Question 18

True or False - fixation at room temperature is sufficient to maintain excellent morphological detail

Answer 18

True

Question 19

Method that slows down decomposition if tissue needs to be photograph and can’t fixed immediately

Answer 19

Refrigeration

Question 19

An organ that continues to undergo mitosis (growth) up to 30 minutes after death (refrigerated)

Answer 19

Bone marrow

Question 19

Example of a cell that deteriorate very quickly?

Answer 19

Brain cells

Question 19

True or False - Nucleic acids do not react with fixatives to any extent at room temperatures, and chemical reactions including those involved in fixation are more rapid at higher temperatures

Answer 19

True

Question 20

True or False - ↑ temperature = ↑ fixation but ↑ autolysis

Answer 20

True

Question 21

True or False - fecal matter and stomach contents can inhibit the penetration of fixative and damage tissue during sectioning, must be removed before fixation

Answer 21

True

Question 21

True or False - tissues can be cut and trimmed without prior fixation, except for the brain which soft when unfixed

Answer 21

True

Question 21

brain is usually suspended whole in 10% buffered formalin for ___ weeks to ensure fixation and some hardening prior to sectioning

Answer 21

2-3 weeks

Question 22

True or False - glutaraldehyde penetrate the best, formalin and alcohol the worst, mercurials and others in between

Answer 22

False - formalin and alcohol penetrate the best, glutaraldehyde the worst, mercurials and others in between

Question 23

Recommended size of the tissue to maintain an adequate fixation time of 4 to 6 hours?

Answer 23

2 cm2 and no more than 4 mm thick

Question 24

True or False - best results are usually obtained using slightly hypertonic solutions (400-450 mOsm) and hypotonic solutions (340 mOsm)

Answer 24

False - best results are usually obtained using slightly hypertonic solutions (400-450 mOsm) and isotonic solutions (340 mOsm)

Question 25

True or False - concentration of fixative should be adjusted down to the lowest level possible

Answer 25

True

Question 25

commonly added to osmium tetroxide fixatives for electron microscopy

Answer 25

sucrose

Question 25

Found to be an ideal concentration for immunoelectron microscopy

Answer 25

low concentrations of glutaraldehyde (0.25%)

Question 25

True or False - the longer the blood supply is interrupted, the poorer the quality of tissue

Answer 25

True

Question 26

True or False - fixatives may act as mordants or accentuators to promote and hasten staining, or they may inhibit certain dyes in favor of another

Answer 26

True

Question 26

________________ intensifies staining while ___________________ inhibits hematoxylin staining

Answer 26

formaldehyde intensifies staining while osmium tetroxide inhibits hematoxylin staining

Question 27

Groups of Fixative that act by crosslinking proteins

Answer 27

Aldehyde and Oxidizing Agents

Question 28

Characteristics of a good fixative causing minimal physical and chemical alteration of the cells and their constituents

Answer 28

it must be isotonic

Question 28

Examples of aldehydes?

Answer 28

formaldehyde, glutaraldehyde

Question 29

Examples of oxidizing agents?

Answer 29

osmium tetroxide, potassium permanganate

Question 30

Group of fixatives that acts as protein denaturing agents?

Answer 30

Alcohol based fixatives

Question 31

True or False - acetic acid is an alcohol based fixative

Answer 31

True

Question 32

Group of fixatives that acts by forming insoluble metallic precipitates

Answer 32

Metallic fixatives

Question 33

Examples of metallic fixatives?

Answer 33

mercuric chloride, picric acid

Question 33

Fixative according to composition that is made up of only one component substance

Answer 33

Simple fixatives

Question 33

Four Major Groups of Fixatives

Answer 33

1. Aldehyde 2. Oxidizing agents 3. Alcohol based 4. Metallic

Question 33

Fixatives according to action that permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question

Answer 33

Microanatomical Fixatives

Question 33

Example of simple fixatives

Answer 33

1. Aldehydes (Formaldehyde, Glutaraldehyde) 2. Metallic Fixatives (Mercuric chloride, Chromate fixatives) 3. Picric acid 4. Acetic acid 5. Acetone 6. Alcohol 7. Osmium Tetroxide

Question 33

Fixative according to composition that is made up of two or more fixatives which have been added together to obtain the optimal combined effect of their individual actions upon the cells and tissue constituents

Answer 33

Compound fixatives

Question 34

Examples of Microanatomical Fixatives

Answer 34

* 10% formal saline * 10% neutral buffered formalin * Heidenhain 's Susa * Formal sublimate (formal corrosive) * Zenker 's solution; Zenker-formal (Kelly 's solution) * Bouin's solution * Brasil's solution

Question 35

Fixatives according to action that preserve specific parts and particular microscopic elements of the cell itself

Answer 35

Cytological Fixatives

Question 36

Nuclear fixatives usually contain _________________as their primary component due to its affinity for nuclear chromatin

Answer 36

glacial acetic acid

Question 37

Nuclear fixatives have a pH of _____

Answer 37

pH of <4.6

Question 38

Example of nuclear fixatives

Answer 38

* Flemming's fluid * Carnoy's fluid * Bouin's fluid * Newcomer's fluid * Heidenhain's Susa * Mercuric chloride

Question 39

Nuclear fixative found to react with viruses and causes the loss of their infective power

Answer 39

Mercuric chloride

Question 40

Cytoplasmic fixative must never contain glacial acetic acid which destroys _____________ and ___________________ of the cytoplasm

Answer 40

Cytoplasmic fixative must never contain glacial acetic acid which destroys mitochondria and Golgi bodies of the cytoplasm

Question 41

Example of cytoplasmic fixative

Answer 41

* Flemming's fluid without acetic acid * Kelly's fluid * Formalin with "post-chroming" * Regaud 's fluid (Muller 's fluid) * Orth 's fluid * ethanol and acetone (precipitant fixatives for RNA, best quantitative results using frozen tissues as the standard)

Question 41

Cytoplasmic fixative have a pH of ___

Answer 41

pH of >4.6

Question 42

Fixative that preserve the chemical constituents of cells and tissues

Answer 42

Histochemical Fixatives

Question 43

Examples of Histochemical Fixatives

Answer 43

* Formal Saline 10% * Absolute Ethyl Alcohol * Acetone * Newcomer's Fluid

Question 43

Form of secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5-3% potassium dichromate for 24 hours to act as mordant for better staining effects and to aid in cytologic preservation of tissues

Answer 43

Post-Chromatization

Question 43

Secondary fixation is done before ____________ and on _________________ before staining, usually with 10% formalin or 10% formol saline as a primary fixative

Answer 43

Secondary fixation is done before dehydration and on deparaffinized sections before staining, usually with 10% formalin or 10% formol saline as a primary fixative

Question 44

Use to remove excess chromates from tissues fixed in Kelly's, Zenker's, and Flemming's solutions

Answer 44

Tap water

Question 45

Used to remove excess formalin

Answer 45

Tap water

Question 46

Used to remove excess osmic acid

Answer 46

Tap water

Question 46

Used to wash out excess amount of picric acid (Bouin's solution)

Answer 46

50-70% alcohol

Question 46

Used to remove excessive mercuric fixatives

Answer 46

Alcoholic iodine

Question 46

Cause of the presence of artefact pigments on tissue sections

Answer 46

Incomplete washing of fixative

Question 46

Cause where tissues are soft and feather-like in consistency

Answer 46

Incomplete fixation

Question 47

Cause of loss or inactivation of enzymes needed for study

Answer 47

wrong choice of fixative

Question 48

Cause of shrinkage and swelling of cells and tissue structure

Answer 48

Overfixation

Question 49

Cause of tissue blocks are brittle and hard

Answer 49

Prolonged fixation

Question 50

well-known artifact that may be produced under acid conditions

Answer 50

Formalin pigment

Question 51

How to reduced formalin pigment?

Answer 51

use of neutral buffered formalin

Question 52

Found in surgical specimens particularly in liver biopsies, associated with an intense eosinophilic staining at the center of the tissue in H&E stained sections

Answer 52

Crush artifact

Question 52

True or False - cryostat or frozen sections should be used in lipid fixation

Answer 52

True

Question 52

Fixative effective for lipids in cryostat sections

Answer 52

mercuric chloride and potassium dichromate

Question 52

Fixative use for phospholipids which contain amino acids

Answer 52

aldehydes

Question 52

Fixative use for unsaturated fatty acids (less lipid can be demonstrated)

Answer 52

formaldehyde

Question 53

Baker's formal-calcium is use to preserve __________________

Answer 53

phospholipids

Question 54

Use in ultrastructural demonstration of cholesterol

Answer 54

digitonin

Question 54

Use in post fixing for ultrastructural demonstration of lipids

Answer 54

imidazole osmium tetroxide

Question 55

True or False - carbohydrates are hydrophobic

Answer 55

carbohydrates are hydrophilic (hold much water in the extracellular space by hydrogen bonding, much water inside cells)

Question 56

Fixative recommended for glycogen

Answer 56

Alcoholic fixatives (Rossman's fluid or cold absolute alcohol)

Question 57

Fixative most commonly used amino acid

Answer 57

neutral buffered formal saline or formaldehyde vapor

Question 58

most useful primary fixatives for electron microscopy (whole procedure performed at 4°C)

Answer 58

osmium tetroxide, glutaraldehyde and paraformaldehyde

Question 59

Fixative use in electron histochemistry and electron immunocytochemistry

Answer 59

Karnovsky's paraformaldehyde-glutaraldehyde

Question 59

Fixative used for fresh frozen cryostat sections for enzyme histochemistry (washed in distilled water prior to enzyme staining)

Answer 59

acetone or formaldehyde

Question 60

Two Classes of Fixation Methods

Answer 60

1. Organic Solvents (alcohols and acetone) 2. Cross-linking Reagents (paraformaldehyde)