PREFINALS: IMPREGNATION AND EMBEDDING

Question 1

AKA Infiltration

Answer 1

IMPREGNATION

Question 2

Removal of clearing agent from the tissue and replacing it with the infiltrating media

Answer 2

IMPREGNATION

Question 3

This infiltrating media will completely fill all tissue cavities, thus giving firm consistency, as well as allow easy handling and cutting of thin tissue

Answer 3

IMPREGNATION

Question 4

Incomplete Impregnation =

Answer 4

Airholes in tissue sections

Question 5

Main factors: method of impregnation, nature and size of tissue, clearing agent used

Answer 5

IMPREGNATION

Question 6

Types of Tissue Impregnation and Embedding Media:

Answer 6

1. Paraffin impregnation
2. Celloidin (Colloidin) impregnation
3. Gelatin impregnation
4. Plastic

Question 7

Simplest, common and best media for routine processing

Answer 7

Paraffin

Question 8

ADV: sections are cut easily without distortion; very rapid (24hrs); permits many staining procedures;

Answer 8

Paraffin

Question 9

DADV: not for fatty tissues; must fully impregnate the tissue to avoid tissue crumbling

Answer 9

Paraffin

Question 10

Paraffin
Melting point for routine work:

Answer 10

56C

Question 11

Paraffin
Never overheat (>60OC):

Answer 11

causes brittleness, shrinkage, hardening; destruction of lymph tissue

Question 12

maintain at 2 to 5C higher than MP of wax

Answer 12

Paraffin oven

Question 12

Wax must be filtered first using coarse filter paper such as Green’s No. 904 in wax oven at 2OC higher than MP of wax

Answer 12

Paraffin

Question 12

Used pure

Answer 12

Paraffin

Question 13

Reusable only once, but remove water first by boiling to 100-105OC

Answer 13

Paraffin

Question 14

Methods of Paraffin Impregnation:

Answer 14

1. Manual
2. Automatic
3. Vacuum Embedding

Question 15

 Atleast 4 changes of paraffin every 15 minutes

Answer 15

Manual

Question 16

Uses machines like Autotechnicon and Elliot Bench-Type Processor, which fixes, dehydrates, clears, and infiltrates tissues

Answer 16

Automatic

Question 17

Infiltration is usually at stations 11 and 12

Answer 17

Autotechnicon

Question 18

ADV: Has constant agitation  speedy procedure

Answer 18

Autotechnicon

Question 19

NOTE: Any odor in clearing agent indicates that the paraffin wax should be changed

Answer 19

Autotechnicon

Question 20

Wax bath thermostat should be set atleast 3 degrees above the MP of paraffin

Answer 20

Autotechnicon

Question 21

Fastest (25-75% reduction of usual impregnation time)

Answer 21

Vacuum Embedding

Question 22

Uses embedding oven with negative atmospheric pressure  rapid removal of air bubbles (e.g. lungs) and clearing agent rapid infiltration

Answer 22

Vacuum Embedding

Question 23

For urgent biopsies, delicate tissues (e.g. CNS, eyes)

Answer 23

Vacuum Embedding

Question 24

Paraffin Wax Substitutes:

Answer 24

1. Paraplast 2. Embeddol 3. Bioloid 4. Tissue Mat 5. Ester Wax 6. Water Soluble Waxes (Polyethylene glycol)

Question 25

Mixture of pure paraffin and synthetic plastic polymer (Dimethyl sulfoxide); more elastic and resilient

Answer 25

Paraplast

Question 26

MP: 56-57C

Answer 26

Paraplast

Question 27

less brittle, and less compressible (MP: 56-57OC)

Answer 27

Embeddol

Question 28

semisynthetic; for embedding of eyes

Answer 28

Bioloid

Question 29

has rubber

Answer 29

Tissue Mat

Question 30

Water insoluble but soluble in 95% ethanol, thus prior clearing is not needed

Answer 30

Ester Wax

Question 31

MP: 46-48C

Answer 31

Ester Wax

Question 32

Harder than paraffin thus used with sliding/sledge-type microtome

Answer 32

Ester Wax

Question 33

But Cellosolve, and xylene may be used if indicated

Answer 33

Ester Wax

Question 34

MP: 38-42OC or 45-56OC

Answer 34

Water Soluble Waxes (Polyethylene glycol)

Question 35

Water Soluble Waxes (Polyethylene glycol) most common:

Answer 35

Carbowax

Question 36

Carbowax absorbs water; no need for dehydration and clearing

Answer 36

Hygroscopic

Question 37

Easily dissolved in water, thus sections are difficult to float out and mount [**Remedy**: add soap to water, or 10% polyethylene glycol 900 in water]

Answer 37

Carbowax

Question 38

Neutral fats and lipids can be demonstrated

Answer 38

Carbowax

Question 39

Not exposed to too much heat, thus for enzyme histochemistry

Answer 39

Carbowax

Question 40

AKA colloidin

Answer 40

Celloidin

Question 41

Purified form of nitrocellulose/gun cotton

Answer 41

Celloidin

Question 42

Concentration: in 2%, 4%, 8% dissolved in equal parts of ether and ROH

Answer 42

Celloidin

Question 42

Specimen with large and hollow cavities which tend to collapse; hard and dense tissues; neurologic tissues

Answer 42

Celloidin

Question 43

ADV: Does not require heat for processing; rubbery

Answer 43

Celloidin

Question 44

DADV: very slow (days to weeks)

Answer 44

Celloidin

Question 45

Celloidin Methods:

Answer 45

1. Wet Celloidin 2. Dry Celloidin

Question 46

For bones, brain, teeth

Answer 46

Wet Celloidin

Question 46

Wet Celloidin procedure:

Answer 46

1. Fixation & Dehydration 2. Place tissue in ether-alcohol 3. Thin celloidin 4. Medium celloidin 5. Thick celloidin 6. Remove specimen and the put it in fresh thick celloidin 7. Keep in jar or dessicator until ether-alcohol evaporates 8. Store tissue block in 70%-80% alcohol

Question 47

No fingerprint marks on surface of tissue block

Answer 47

Complete impregnation

Question 48

For whole eye sections

Answer 48

Dry Celloidin

Question 48

Uses Gilson’s mixture (equal parts of chloroform and cedar wood oil) for storage, instead of 70% to 80% alcohol

Answer 48

Dry Celloidin

Question 49

Has lower viscosity, thus can be used in high concentrations, and rapid tissue penetration

Answer 49

Nitrocellulose Method/Low Viscosity Nitrocellulose (LVN)

Question 50

# * ADV: Harder tissue block, thus thinner sections are possible

Answer 50

Nitrocellulose Method/Low Viscosity Nitrocellulose (LVN)

Question 51

DADV: Explosive when dry d/t nitrates

Answer 51

Nitrocellulose Method/Low Viscosity Nitrocellulose (LVN)

Question 52

Nitrocellulose Method/Low Viscosity Nitrocellulose (LVN) (e.g. oleum ricini or castor oil) is needed to prevent tissue cracking in chrome-mordanted tissues

Answer 52

Plasticizer

Question 53

Rarely used

Answer 53

Gelatin

Question 54

For histochemical, enzyme studies, and frozen sections

Answer 54

Gelatin

Question 55

ADV: Water soluble (no dehydration and clearing needed)

Answer 55

Gelatin

Question 56

DADV: may decay

Answer 56

Gelatin

Question 57

Gelatin TSE must be:

Answer 57

<2-3mm thick

Question 57

Gelatin Procedure:

Answer 57

1. Wash out of fixative 2. Put tissue in 10% gelatin with 1% phenol 3. 20% gelatin with 1% phenol 4. Fresh 20% gelatin with 1% phenol 5. Cool in refrigerator 6. 10% formalin

Question 58

Gelatin TSE:IMPERGNATING AGENT ratio is set at

Answer 58

1:25

Question 59

Gelatin must be added to prevent molds

Answer 59

1% Phenol

Question 60

AKA Casting, Blocking

Answer 60

EMBEDDING

Question 61

Placing the impregnated tissue into a mold with embedding media, and then allowing the media to solidify

Answer 61

EMBEDDING

Question 62

Arrangement of the tissue in a precise position in the mold during embedding

Answer 62

Orientation

Question 63

Surface to be cut should be parallel to bottom of the mold

Answer 63

EMBEDDING

Question 64

Molds should bear the accession number

Answer 64

EMBEDDING

Question 65

EMBEDDING Procedure:

Answer 65

Put tissue with label on a mold, immerse them in melted paraffin at 5-10 OC higher than MP of wax, then rapidly cool in a refrigerator at -5 OC, or in cold water

Question 66

Embedding Molds:

Answer 66

1. Leuckhart’s Embedding mold 2. Compound embedding unit 3. Plastic embedding rings and base molds 4. Disposable molds

Question 67

L-shaped strips of heavy brass and metal; reusable and adjustable

Answer 67

Leuckhart’s Embedding mold

Question 68

Interlocking plates resting on flat metal base; for batch embedding

Answer 68

Compound embedding unit

Question 69

Plastic embedding rings and base molds

Answer 69

Special stainless steel base mold fitted with a plastic embedding ring (serves as block holder during cutting)

Question 70

has paraffin reservoir, tissue tank, warm plate and cold plate (-5C)

Answer 70

TissueTek

Question 71

(thin plastic mold)

Answer 71

Peel-away

Question 72

Disposable molds:

Answer 72

1. Peel-away 2. Plastic Ice tray 3. Paper boats

Question 73

(inner mold smeared with glycerin or liquid paraffin),

Answer 73

Plastic Ice tray

Question 74

Other Embedding Methods:

Answer 74

1. Double embedding method 2. Plastic (Resin) embedding

Question 75

celloidin first, then paraffin; for large blocks of dense tissues; obsolete

Answer 75

Double embedding method

Question 76

Plastic (Resin) embedding Classes:

Answer 76

* Epoxy * Polyester * Acrylic Plastics

Question 76

For High resolution light microscopy of thinner than usual sections, renal biopsies, BM biopsies

Answer 76

Plastic (Resin) embedding

Question 76

For electron microscopy

Answer 76

Epoxy

Question 77

Most widely applied, but carcinogenic due to vinylcyclohexane dioxide (VCD) component

Answer 77

Epoxy

Question 77

Epoxy Types:

Answer 77

* Bisphenol A (Araldite) * Glycerol (Epon) * Cyclohexene Dioxide (Spurr)

Question 78

slow

Answer 78

Bisphenol A (Araldite)

Question 79

low viscosity

Answer 79

Glycerol (Epon)

Question 80

very low viscosity; fastest

Answer 80

Cyclohexene Dioxide (Spurr)

Question 80

For electron microscopy; seldom used

Answer 80

Polyester

Question 81

For High resolution light microscopy

Answer 81

Acrylic Plastics

Question 82

E.g., polyglycol methacrylate (GMA), methyl metacrylate (MMA)

Answer 82

Acrylic Plastics

Question 83

catalyst; forms radicals, which are site for polymerization

Answer 83

Benzoyl peroxide

Question 84

Acrylic plastics must be stored in dark bottles to prevent radical formation and premature polymerization

Answer 84

Acrylic Plastics

Question 85

Embedding media may be stained, thus use hydrophobic MMA

Answer 85

Acrylic Plastics