Fixation

Question 1

What are the functions of a fixative

Answer 1

prevent autolysis, stabilize tissue morphology and enhance staining

Question 2

What are the two major ways that fixatives alter tissue proteins

Answer 2

denaturation and formation of crosslinks

Question 3

What are the four methods of denaturation

Answer 3

heat, alcohols, acids and heavy metals

Question 4

What is the most common kind of cross-linking fixatives

Answer 4

aldehydes

Question 5

What do aldehydes bind to

Answer 5

amino groups as well as lysine, cysteine, serine and threonine residues

Question 6

How does fixation using oxidizing agents work

Answer 6

poorly understood but it is known that tissues are stabilized by cross-linking reactive groups in close prozimity

Question 7

What are coagulant fixatives

Answer 7

cause distortion and destruction of fragile cytoplasmic constituents but support proteins. They work quickly

Question 8

What are examples of coagulant fixatives

Answer 8

alcohols, acetone, acid fixatives and metal salts

Question 9

What are non-coagulant fixatives

Answer 9

stabilize tissue morphology by forming bridges between adjacent reactive areas within proteins. Slower

Question 10

What are examples of non-coagulant fixatives

Answer 10

aldehydes and oxidizing reagents

Question 11

What is an additive

Answer 11

chemically binds with the proteins or other tissue component

Question 12

What is a non-additive

Answer 12

disrupting tissue structure without becoming chemically incorporated

Question 13

What are the main factors that affect fixation

Answer 13

temperature, size/thickness/tissue type, time, volume of fixative

Question 14

What is the minimum fixation time

Answer 14

8 hours

Question 15

What is 100% formalin

Answer 15

47-40% formaldehyde in water

Question 16

What is neutral buffered formalin

Answer 16

10% formalin in water

Question 17

How much formaldehyde is in neutral buffered formalin

Answer 17

3.7-4%

Question 18

What is paraformaldehyde

Answer 18

a white powder that precipitates out of concentrated formaldehyde solutions

Question 19

What type of fixative is formalin

Answer 19

additive, non-coagulant, cross-linking

Question 20

What are the pros and cons of formalin

Answer 20

less shrinkage but hardens tissue

Question 21

What is a limit of glutaraldehyde

Answer 21

tissue must be very thin

Question 22

What type of fixative is glutaraldehyde

Answer 22

additive, non-coagulant fixative

Question 23

When is glutaraldehyde used

Answer 23

for electron microscopy

Question 24

Why is acetic acid added to fixatives

Answer 24

it preserves nucleoproteins and precipitates DNA, it can lyse RBCs and it swells tissues

Question 25

What is picric acid

Answer 25

both a fixative and a stain

Question 26

What type of fixative is picric acid

Answer 26

additive coagulant

Question 27

Why must picric acid be avoided if DNA/RNA analysis is anticipated

Answer 27

it hydrolyzes nucleic acids

Question 28

What type of dyes does picric acid enhance

Answer 28

anionic dyes

Question 29

What must occur after fixation with picric acid

Answer 29

the picric acid must be neutralized

Question 30

What is ethanol used for in the histo lab

Answer 30

mostly as dehydrating agents

Question 31

What does fixation with alcohols do to tissues

Answer 31

leaves tissue hard and shrunken

Question 32

What is ethanol notable for

Answer 32

preserving glycogen and urate crystals

Question 33

What type of fixative is osmium tetroxide

Answer 33

additive, non-coagulant

Question 34

What is osmium tetroxide used for

Answer 34

electron microscopy

Question 35

What makes osmium tetroxide notable

Answer 35

it can fix lipids causing them to appear black

Question 36

What fixatives are included in bouins solution

Answer 36

formaldehyde, acetic acid and aqueous picric acid

Question 37

What makes bouins solution ideal

Answer 37

good for trichromes as the picric acid enhances staining, the acetic acid helps counteract shrinkage, and picric acid leaves the block soft and easy to section

Question 38

Why is it important to wash tissue after fixation with bouins solution

Answer 38

the residual picric acid will digest the section over time

Question 39

What are the components of B5

Answer 39

mercuric chloride, sodium acetate, formalin and water

Question 40

What is B5 most often used

Answer 40

hematopeitic and luphoreticular tissues

Question 41

Why must tissues be stored in 70% alcohol after fixation with B5

Answer 41

it is not tolerant

Question 42

What is B-plus

Answer 42

a modern version of B5 that uses zinc chloride instead of mercuric chloride

Question 43

What are the components of Clarke's fluid

Answer 43

ethanol and glacial acetic acid

Question 44

What does Clarke's fluid do

Answer 44

preserves nucleic acids, extracts lipids and maintains microanatomical structures

Question 45

What is the storage solution after fixation with Clarke's fluid

Answer 45

95% ethanol

Question 46

What are the components of modified formalin

Answer 46

formaldehyde and alcohol

Question 47

What is the benefit of modified formalin

Answer 47

it speeds up tissue fixation and begins dehydration

Question 48

What does adding zinc sulfate to NBF do

Answer 48

preserves tissue antigenicity with prolonged storage and improves nuclear detail

Question 49

What is proprietary formalin-free fixative

Answer 49

usually contain an alcohol and other additives, safer solution than formalin

Question 50

What is melanin

Answer 50

a dark brown, granular pigment often demonstrated using the fontana-masson stain and may be removed using potassium permanganate

Question 51

What is formalin pigment

Answer 51

amorphous birefringent dark brown or black pigment formed from hemoglobin reacting with formaldehyde in acidic conditions

Question 52

How to remove formalin pigment from a section

Answer 52

saturated solution of picric acid and alcohol followed by a running water wash

Question 53

What is mercury pigment

Answer 53

a crystalline birefringent brown pigment

Question 54

How to correct mercury pigment

Answer 54

use a iodine treatment followed by a sodium thiosulphate treatment

Question 55

What is picric acid pigment

Answer 55

stains tissue components an intense yellow colour, rinising the tissue helps remove it

Question 56

What are two examples of exogenous pigments

Answer 56

carbon and tattoo ink