Fixatives

Question 1

What is the difference between tolerant and non tolerant?

Answer 1

Tolerant:
- Tissues can be exposed for a long time without adverse effects.
Non-Tolerant:
- Tissues cannot be exposed for a long time without adverse effects.

Question 2

What is a primary fixing agent?

Answer 2

A single chemical that has the ability to denature protein

Question 3

How are primary fixatives typically used?

Answer 3

Almost never used alone; usually combined in a compound fixative

Question 4

What are some common primary fixatives?

Answer 4

• Formaldehyde
• Potassium dichromate
• Mercuric chloride
• Ethanol
• Picric acid
• Acetic acid
• Osmium tetroxide
• Glutaraldehyde

Question 5

What is formaldehyde commonly known as?

Answer 5

The universal fixative

Question 6

What is the typical concentration of formalin used as a routine fixative?

Answer 6

10% vol/vol, which contains 4% formaldehyde

Question 7

What type of fixative is formaldehyde considered?

Answer 7

An additive fixing agent

Question 8

What is the main reaction site for formaldehyde in tissues?

Answer 8

Amino group (NH2) found on the side chains of amino acids

Question 9

What is formed when formaldehyde reacts with tissue proteins?

Answer 9

A cross-linkage called a methylene bridge

Question 10

True or False: Formaldehyde causes more shrinkage than any other fixative.

Answer 10

False

Question 11

What happens to glycogen when fixed with formaldehyde?

Answer 11

Glycogen is indirectly fixed; trapped in a meshwork

Question 12

What effect does formaldehyde have on lipids?

Answer 12

Lipids are preserved but not made insoluble

Question 13

How can formalin pigment be formed?

Answer 13

When formaldehyde is used in acidic solutions (pH < 5)

Question 14

What happens to glycogen when fixed with potassium dichromate?

Answer 14

Glycogen is oxidized and not readily demonstrable

Question 15

What is the effect of potassium dichromate on tissue pH?

Answer 15

It is pH dependent; non-coagulating at pH > 3.8

Question 16

What is a disadvantage of potassium dichromate?

Answer 16

Highly toxic and considered a carcinogen

Question 17

What is mercuric chloride known for?

Answer 17

Powerful protein coagulant and extremely toxic

Question 18

How does mercuric chloride affect lipids?

Answer 18

Has no reaction with lipids; does not fix or destroy them

Question 19

What must be done to remove mercury pigment from tissues?

Answer 19

Treat with metallic iodine followed by sodium thiosulfate

Question 20

What effect does mercuric chloride have on tissues if over-exposed?

Answer 20

Excessive hardening, making tissues brittle

Question 21

What is the mode of action of mercuric chloride?

Answer 21

Combines with acid groups of proteins and inhibits freezing

Question 22

What is the procedure for mercury pigment removal from tissue?

Answer 22

1. Immerse in alcoholic iodine for 3 minutes.
2. Wash in running tap water for 2 minutes.
3. Immerse in 5% HYPO.
4. Wash in running tap water for 2 minutes.
5. Stain as required.

This procedure effectively removes mercury and iodine discolouration.

Question 23

What is a key characteristic of mercuric chloride in histological applications?

Answer 23

It is never used alone and always combined with gentler solutions.

Mercuric chloride is typically part of compound fixatives.

Question 24

True or False: Ethanol is a good fixing agent for demonstrating microanatomy.

Answer 24

False

Ethanol causes extreme shrinkage, making it a poor choice for microanatomy demonstration.

Question 25

What does ethanol best preserve?

Answer 25

Glycogen ## Footnote Ethanol precipitates glycogen, allowing it to be demonstrated in tissue sections.

Question 26

What type of fixative is picric acid classified as?

Answer 26

Additive coagulant fixative ## Footnote Picric acid forms additive compounds with proteins.

Question 27

What is a major disadvantage of picric acid?

Answer 27

Causes extreme shrinkage and penetrates poorly ## Footnote These properties limit its effectiveness in some applications.

Question 28

How does acetic acid affect the nuclei during fixation?

Answer 28

Coagulating and intolerant ## Footnote Acetic acid causes significant swelling and affects nucleoproteins.

Question 29

What is the main use of osmium tetroxide in histology?

Answer 29

Fixation for electron microscopy ## Footnote Osmium tetroxide preserves fine cellular structures.

Question 30

What type of fixative is glutaraldehyde?

Answer 30

Non-coagulating additive fixative ## Footnote Glutaraldehyde crosslinks proteins and is used primarily for electron microscopy.

Question 31

What is a disadvantage of glutaraldehyde?

Answer 31

Slow penetration and must be stored in a refrigerator ## Footnote These factors affect its usability in tissue processing.

Question 32

Fill in the blank: Picric acid is used as both a _______ and a dye.

Answer 32

fixative ## Footnote Picric acid serves dual purposes in histological applications.

Question 33

True or False: Acetic acid preserves lipids in tissues.

Answer 33

False ## Footnote Acetic acid does not fix or destroy lipids; they are removed during processing.

Question 34

What happens to glycogen when fixed with glutaraldehyde?

Answer 34

Glycogen proteins are precipitated in a meshwork ## Footnote This indirect fixation allows glycogen to be demonstrated with specific stains.

Question 35

What is glyoxal used for in histology?

Answer 35

Replacing glutaraldehyde and formaldehyde ## Footnote Glyoxal is less toxic and does not give off vapors.

Question 36

What are the primary fixing agents used in compound fixatives?

Answer 36

* Neutral buffered formalin * Formal saline * Formal calcium * Buffered formal sucrose * Formal alcohol * Zenker’s * Bouin’s * B – 5 * Zinc containing

Question 37

What is the purpose of combining fixing agents in a compound fixative?

Answer 37

To balance/counteract the harmful effects of one fixative with the beneficial effects of another.

Question 38

Which agent is commonly used to counteract shrinkage in a compound fixative?

Answer 38

Acetic acid.

Question 39

What is the function of salts or buffers in a compound fixative?

Answer 39

* Maintain osmotic pressure * Aid in penetration of fixative into tissues.

Question 40

What are the categories into which Compound Fixatives are subdivided?

Answer 40

* Microanatomical * Cytological * Histochemical.

Question 41

What are the components of Neutral Buffered Formalin?

Answer 41

* Formaldehyde * Phosphate buffer * Water.

Question 42

What is a disadvantage of Neutral Buffered Formalin?

Answer 42

Gradual loss of basophilic staining of cytoplasm and nucleus after prolonged storage.

Question 43

What is the stock solution of Zenker’s Fluid composed of?

Answer 43

* Mercuric chloride * Potassium dichromate * Sodium sulphate * Distilled water * Glacial acetic acid.

Question 44

What is a disadvantage of Zenker’s Acetic Fixative?

Answer 44

Turns muddy brown after 24 hours.

Question 45

What is the purpose of acetic acid in Zenker’s Acetic Fixative?

Answer 45

Acts as a primary fixative and excellent nucleoprotein fixative, counteracts shrinkage.

Question 46

What are the components of Bouin’s Fixative?

Answer 46

* Saturated picric acid * Formalin * Glacial acetic acid.

Question 47

What is the primary use of Bouin’s Fixative?

Answer 47

Good general all-purpose fixative, excellent for preserving soft and delicate structures.

Question 48

What are the disadvantages of Bouin’s Fixative?

Answer 48

* Lyses RBCs * Alters and decreases lipids * Tissues must be transferred directly to alcohol to prevent loss of water soluble picrate.

Question 49

What are the ingredients of B – 5 Fixative?

Answer 49

* Mercuric chloride * Sodium acetate * Distilled water * Formalin (added immediately prior to use).

Question 50

What tissues is B – 5 Fixative used to fix?

Answer 50

Hematopoietic and lymphoreticular tissues.

Question 51

What are the components of Z – 5 Fixative?

Answer 51

* Zinc sulphate * Unbuffered formalin * Ethanol.

Question 52

What is the composition of Carnoy’s Fixative?

Answer 52

* Absolute ethanol * Chloroform * Glacial acetic acid.

Question 53

What is the unique feature of Carnoy’s Fixative?

Answer 53

The only compound fixative classified as a cytological fixative.

Question 54

How quickly does Carnoy’s Fixative penetrate tissue?

Answer 54

Rapid penetration for the first 2mm of tissue.

Question 55

What type of fixative is Carnoy’s classified as?

Answer 55

Carnoy’s is classified as a cytological fixative.

Question 56

How long does it take for a 2mm tissue to be fixed using Carnoy’s?

Answer 56

A 2mm tissue will be fixed in 15 minutes.

Question 57

What advantage does Carnoy’s fixation provide for urgent diagnosis?

Answer 57

Rapid fixation at room temperature allows for STAT or urgent diagnosis.

Question 58

What components does Carnoy’s fluid excellently preserve?

Answer 58

Carnoy’s gives excellent nuclear fixation and preserves nucleic acids.

Question 59

What substance is an important ingredient in fixing glycogen?

Answer 59

Alcohol is an important ingredient to fix glycogen.

Question 60

What are the disadvantages of using Carnoy’s fluid?

Answer 60

Carnoy’s fluid causes considerable shrinkage and destroys or dissolves most cytoplasmic elements.

Question 61

What happens to lipids when using Carnoy’s fluid?

Answer 61

Lipids are dissolved by ethanol.

Question 62

What effect does acetic acid have on RBCs in Carnoy’s fluid?

Answer 62

RBCs are lysed by acetic acid.

Question 63

What is the purpose of histochemical fixatives?

Answer 63

Histochemical fixatives preserve chemical constituents in or on cells.

Question 64

What is often lost when using histochemical fixatives?

Answer 64

The architecture of the tissue and cellular morphology are often forfeited.

Question 65

What is a secondary fixative?

Answer 65

A secondary fixative is an additional fixative used after the first fixative.

Question 66

What improvement does secondary fixation provide?

Answer 66

Shows general improvement in preservation of tissue.

Question 67

What is the primary fixative usually utilized?

Answer 67

NBF (Neutral Buffered Formalin).

Question 68

What are common components of secondary fixatives?

Answer 68

* Mercury * Picric acid

Question 69

What is the purpose of mordanting qualities in fixatives?

Answer 69

Mordanting properties enhance or improve subsequent staining of the tissue.

Question 70

What is mercuric chloride used for?

Answer 70

Often used as a secondary fixative.

Question 71

What is Bouin’s solution used for?

Answer 71

Used as a mordant prior to staining tissue sections with the trichrome staining method.

Question 72

What is the recommended ratio of fixative to tissue for proper fixation?

Answer 72

20:1.

Question 73

What can result from inadequate fixative volume?

Answer 73

Poor fixation.