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MTLE > Histopathologic Techniques > Flashcards

Histopathologic Techniques Flashcards

1
Q

Steps in Tissue processing

A

Fixation
Dehydration
Clearing
Infiltration
Embedding
Trimming
Sectioning
Staining
Mounting
Labelling

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2
Q

first and most important step

A

fixation

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3
Q

two purpose of fixation

A
  1. Preserve the morphological and chemical integrity of cell
  2. Harden and protect tissue for further handling
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4
Q

primary purpose of fixation

A

preserve morphological and chemical integrity

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5
Q

tissues must be fixed _____

A

within 1hr to prevent putrefaction and autolysis

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6
Q

penetration rate of formalin

A

1mm/hr

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7
Q

ratio of fixative to tissue (routine)

A

20:1

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8
Q

ratio of fixative to tissue (Oste)

A

5-10 x

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9
Q

ratio of fixative to tissue (museum preparations)

A

≥50 x

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10
Q

if not fixed ASAP, autopsy materials must be:

A
  1. placed in mortuary ref at 4C
  2. undergo arterial embalming
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11
Q

if not fixed ASAP, surgical specimens must be:

A

refrigerated, but do not freeze

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12
Q

must be fixed before grossing (suspended whole in 10% NBF for 2-3 weeks)

A

brain

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13
Q

tissues tend to float (put in cotton or open completely before fixation)

A

hollow organs (stomach, intestines)

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14
Q

tissues tend to float (cover with gauze)

A

air-filled lungs

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15
Q

fixed (injection with formol alcohol) before grossing, then immerse in fixative

A

eyes

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16
Q

water must not be used in tissues with _____ , because it is water-soluble

A

glycogen

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17
Q

must be washed with water overnight, then immerse in tissue softeners

A

hard tissues

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18
Q

example of hard tissues:

A

cervix
fibroids
hyperkeratotic skin
fingernails

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19
Q

factors involved in fixation:

pH

A

6 - 8

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20
Q

factors involved in fixation:

temperature
*routine: _____
*autotechnicon: _____
*EM and histochem: _____
*rapid fixation: _____
*tissues with TB: _____

A

room temperature
40C
0-4C
60C
100C

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21
Q

factors involved in fixation:

size of tissue
*routine (LM): _____
*edematous lungs: _____
*EM: _____

A

2cm^2 (<5mm)
1 - 2 cm thick
1 - 2 mm^2

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22
Q

factors involved in fixation:

osmolality

A

slightly hypertonic
(400-450 mOsm)

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23
Q

factors that accelerates/hastens fixation

A
  1. smaller tissues
  2. heat (37-56C)
  3. agitation
  4. vacuum
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24
Q

factors that retards/slows down fixation

A
  1. larger tissues
  2. cold temperature
  3. mucus & blood (flush w/NSS)
  4. fatty tissues (slice thinly)
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25
Q

mechanism of fixative that becomes part of the tissue; form cross-links/complexes; example is aldehyde fixatives

A

additive fixatives

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26
Q

mechanism of fixative that does not become part of the tissue; removes bound water; example is alcohol fixatives

A

non-additive fixatives

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27
Q

composition of fixative where there is only 1 fixative

A

simple fixative

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28
Q

composition of fixative where there are 2 or more fixatives

A

compound fixative

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29
Q

action of fixative that permits the microscopic study of tissue without structural pattern and intercellular relationship alteration

A

microanatomical

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30
Q

action of fixative that preserves specific cell parts/elements at the expense of other cellular components

A

cytological

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31
Q

categories of cytological fixatives:

A

nuclear
cytoplasmic
histochemical

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32
Q

preserves nucleus and chromatin material

A

nuclear fixative

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33
Q

preserves cytoplasm and membrane-bound organelles

A

cytoplasmic fixative

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34
Q

preserves chemical components

A

histochemical fixative

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35
Q

example of nuclear fixative:

A

Heidenhain’s
Newcomer’s
Bouin’s
Flemming’s with acetic acid
Carnoy’s

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36
Q

example of cytoplasmic fixative:

A

Helly’s
Orth’s
Regaud’s
Flemmings w/o acetic acid
Formalin w/ post chroming

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37
Q

example of histochemical fixative:

A

Absolute ethanol
Newcomer’s
Acetone
10% Formol saline

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38
Q

example of microanatomical fixative:

A

Heidenhain’s
Brasil’s
Bouin’s
10% Formol saline
10% NBF
Zenker’s
Formol sublimate

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39
Q

acid fixative fixes _____
basic fixative fixes ______

acid stain stains _____
basic stain stains _____

A

nucleus
cytoplasm

cytoplasm
nucleus

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40
Q

List of fixatives:

A
  1. aldehyde
  2. metallic
  3. alcoholic
  4. picric acid
  5. glacial acetic acid
  6. osmium tetroxide
  7. trichloroacetic acid
  8. acetone
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41
Q

aldehyde fixatives:

A
  1. formalin/formaldehyde
  2. 10% NBF
  3. 10% Formol saline
  4. formol corrosive
  5. formol calcium
  6. Gendre’s (alcoholic formalin)
  7. glutaraldehyde
  8. 4% paraformaldehyde
  9. acrolein
  10. glyoxal
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42
Q

most commonly used fixative

A

10% formalin

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43
Q

problems using formalin:

A
  1. decomposition to formic acid
  2. prolonged storage produces white precipitate
  3. brown/black crystalline precipitates on blood-containing tissues
  4. fumes are irritating to the eyes, nose, and skin
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44
Q

routine and best general tissue fixative

A

10% NBF

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45
Q

best fixative for iron-containing pigments and elastic fibers

A

10% NBF

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46
Q

CNS and postmortem fixative

A

10% formol saline

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47
Q

fixative ideal for silver impregnation

A

10% formol saline

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48
Q

_____ concentration of formol saline for enzyme histochemistry

A

4%

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49
Q

composed of formalin + mercuric chloride

A

formol corrosive

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50
Q

ideal fixative for silver reticulin methods

A

formol corrosive

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51
Q

fixative for lipids in frozen sections

A

formol calcium

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52
Q

formalin + 95% ethanol + picric acid + glacial acetic acid

A

Gendre’s (alcoholic formalin)

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53
Q

a sputum fixative, but is also good for glycogen and microincineration studies

A

Gendre’s

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54
Q

primary fixative for transmission electron microscopy

A

Glutaraldehyde

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55
Q

a formalin polymer in white powder form; also for EM

A

4% paraformaldehyde

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56
Q

mixture of glutaraldehyde and paraformaldehyde

A

acrolein

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57
Q

smallest aldehyde

A

glyoxal

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58
Q

metallic fixatives:

A
  1. mercuric chloride
  2. chromate
  3. lead
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59
Q

fixative of choice for cell detail preservation in tissue photography

A

mercuric chloride

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60
Q

fixative recommended for renal biopsies

A

mercuric chloride

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61
Q

may produce black deposits except Heidenhain’s susa

A

mercuric chloride

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62
Q

to remove black deposits, do _____

A

dezenkerization

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63
Q

mercuric chloride _____ tissues

glacial acetic acid _____ tissues

A

shrinks
swells

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64
Q

examples of mercuric chloride fixative:

A
  1. B5
  2. Ohlmacher’s
  3. Schauddin’s
  4. Carnoy-Lebrun’s
  5. Heidenhain’s SuSa
  6. Zenker
  7. Zenker-formol (Helly’s)
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65
Q

alternative for mercuric chloride

A

zinc sulfate

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66
Q

bone marrow fixative w/ anhydrous sodium acetate

A

B5

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67
Q

rapid fixative that gives excellent nuclear differentiation

A

Ohlmacher’s
Carnoy-Lebrun’s

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68
Q

fixative for stool

A

Schaudinn’s

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69
Q

fixative for skin biopsies

A

Heidenhain’s SuSa

Su= sublimat
Sa= saure

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70
Q

fixative for trichrome staining and bone marrow (recommended)

A

zenker

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71
Q

fixative for pituitary glands, intercalated disks, and BM w/ potassium dichromate

A

zenker-formol (Helly’s)

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72
Q

fixative that has a fine, yellow-brown pigment (removed w/ acid alcohol)

A

chromate

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73
Q

example of chromate fixatives:

A
  1. chromic acid
  2. Regaud’s (Moller’s)
  3. Orth’s
  4. Potassium dichromate
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74
Q

fixative for carbohydrates

A

chromic acid

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75
Q

fixative for chromatin, mitotic figures and mitochondria

A

Regaud’s (Moller’s)

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76
Q

it demonstrates rickettsia, tissue necrosis and early degenerative processes

A

Orth’s

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77
Q

fixative mainly for mitochondria; if acidified, it destroys mitochondria but fixes cytoplasm, chromatin and chromosomes

A

potassium dichromate

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78
Q

fixes mucin and mucopolysaccharides

A

lead

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79
Q

acts as fixative and dehydrating agents

A

alcoholic fixatives
acetone

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80
Q

excellent preservative for glycogen but rapidly denatures proteins

A

alcoholic fixatives

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81
Q

preserves nuclear stans but dissolves fats

A

alcoholic fixatives

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82
Q

example of alcoholic fixatives:

A
  1. methanol
  2. ethanol
  3. isopropyl alcohol
  4. carnoy’s fluid
  5. newcomer’s
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83
Q

fixative for blood and BM smears

A

methanol

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84
Q

preserves but does not fix glycogen; useful for PCR

A

ethanol

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85
Q

fixative for touch preparations and Wright-Giemsa stain

A

isopropyl alcohol

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86
Q

most rapid fixative

A

carnoy’s fluid

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87
Q

for fixing chromosomes and urgent biopsies

A

carnoy’s fluid

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88
Q

fixative for mucopolysaccharides and nuclear proteins

A

newcomer’s

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89
Q

highly explosive fixative when dry

A

picric acid

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90
Q

an excellent glycogen fixative

A

picric acid

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91
Q

small tissue fragments can be seen

A

picric acid

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92
Q

major disadvantage: excessive yellow staining

A

picric acid

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93
Q

example of picric acid fixatives:

A
  1. bouin’s
  2. brasil’s
  3. hollande’s
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94
Q

fixative for embryos, pituitary and Masson’s trichrome stain; not for kidney biopsies

A

bouin’s

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95
Q

less messy than bouin’s

A

brasil’s

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96
Q

less lysis than bouin’s, with decalcifying properties

A

hollande’s

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97
Q

fixative for GI tract and endocrine tissues

A

hollande’s

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98
Q

most commonly combined with other fixatives; solidifies at 17C

A

glacial acetic acid

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99
Q

fixes and precipitates nucleoproteins, chromosomes, and chromatin

A

glacial acetic acid

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100
Q

pale yellow powder that dissolves in water

A

osmium tetroxide

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101
Q

excellent lipid fixative

A

osmium tetroxide

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102
Q

fixes myelin and peripheral nerves

A

osmium tetroxide

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103
Q

secondary fixative for EM

A

osmium tetroxide

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104
Q

has a black precipitate crystal pigment (remove with cold water)

A

osmium tetroxide

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105
Q

fixative that does not use hematoxylin, because it inhibits hematoxylin

A

osmium tetroxide

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106
Q

most common chrome-osmium acetic acid fixative for nuclear structures

A

Flemming’s with acetic acid

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107
Q

OsTe fixative for cytoplasmic structures, especially mitochondria

A

flemming’s w/o acetic acid

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108
Q

fixative and weak decalcifying agent

A

trichloroacetic acid

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109
Q

precipitates protein with a swelling effect that counteracts shrinkage of other fixatives

A

trichloroacetic acid

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110
Q

fixative that has a softening effect on dense tissues

A

trichloroacetic acid

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111
Q

fixative used at ice-cold temperatures (-5 to 4C)

A

acetone

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112
Q

fixative for h2o-diffusable enzymes (phosphatases and lipases) and rabies diagnosis

A

acetone

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113
Q

fixation for bacteriologic smears and frozen sections

A

heat fixation

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114
Q

mechanism of heat fixation

A

thermal coagulation of tissue proteins

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115
Q

technique that can penetrate tissues with 10-15 um thickness with an optimum temperature of 45-55C; it increases movement of molecules and accelerates tissue processing stages

A

microwave technique

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116
Q

a rapid microwave fixative that has a mixture of methanol and polyethylene glycol (PEG); it can recover DNA, RNA and proteins for molecular analysis

A

Universal Molecular Fixative (UMFIX)

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117
Q

fixative for enzyme histochemistry:

A
  1. 4% formalin (formol saline)
  2. acetone (cryostat sections)
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118
Q

fixative for electron microscopy:

A
  1. glutaraldehyde
  2. osmium tetroxide
  3. paraformaldehyde
  4. zamboni’s (immunohistochem)
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119
Q

fixative for electron immunocytochemistry:

A
  1. karnovsky’s paraformaldehyde-glutaraldehyde
  2. acrolein
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120
Q

an already fixed tissue is placed into another fixative

A

secondary fixation

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121
Q

an already fixed tissue is fixed using 2.5-3% potassium dichromate (mordant) for one day

A

post-chromatization (post-mordanting)

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122
Q

to remove excess fixative, wash out with:

  1. _____: formalin, chromates. osmic acid
  2. _____: picric acid (bouin’s)
  3. _____: mercuric chloride fixatives
A

tap water
50-70% alcohol
alcoholic iodine

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123
Q

transport medium for unfixed tissues (renal, skin, oral mucosa); not a fixative; rather, refrigerated

A

Michel’s solution

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124
Q

done after fixation and before dehydration (optional)

A

decalcification

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125
Q

it is done to remove calcium or lime salts from bones or calcified tissue

A

decalcification

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126
Q

ratio of decalcifier to tissue

A

20:1

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127
Q

optimum temperature in decalcification:

*routine
*impairs Van Gieson nuclear staining
tissue digestion

A

room temperature (18-30C)
37C
55C

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128
Q

duration of decalcification

A

1-2 days

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129
Q

types of decalcifying agents:

A
  1. acids
  2. chelating agents
  3. ion exchange resin
  4. electrophoresis
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130
Q

example of acid decalcifying agents:

A
  1. 5-10% nitric acid
  2. HCl
  3. 5% formic acid
  4. TCA
  5. sulfurous acid
  6. chromic acid (flemming’s)
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131
Q

most common decalcifying agent

A

5-10% nitric acid

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132
Q

type of nitric acid that contains formalin

A

formol nitric acid

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133
Q

type of nitric acid that is also a tissue softener

A

perenyi’s fluid

134
Q

considered as the most rapid decalcifying agent

A

phloroglucin nitric acid

135
Q

disadvantage of nitric acid

A

imparts a yellow color due to nitrous acid

136
Q

remedy for yellow color of nitric acid

A

5% sodium thiosulfate or urea crystals

137
Q

for slower decalcification of block; slower and produces more distortion to tissues

A

HCl

138
Q

contains 36% HCl or 36% NaCl; used for teeth and small bone pieces

A

Von Ebner’s fluid

139
Q

a fixative and moderate acting decalcifier

A

5% formic acid

140
Q

best general decalcifying agent, can also be used for teeth and small bone pieces

A

5% formic acid

141
Q

decalcifier of post-mortem research tissues and suitable for immunohistochemical staining

A

5% formic acid

142
Q

has a better nuclear staining than nitric acid; for BM and cartilage

A

formic acid-sodium citrate solution

143
Q

a fixative and weak decalcifying agent which permits good nuclear staining but slow; only for small bone spicules

A

TCA

144
Q

weak decalcifying agent; only for minute bone pieces

A

sulfurous acid

145
Q

also for minute bone pieces; carcinogenic

A

chromic acid (Flemming’s)

146
Q

it is combined with calcium and other salts to form complexes pH is adjusted to 7-7.4

A

chelating agents

147
Q

excellent for IHC and EM, but inactivates alkaline phosphatase (remedy: magnesium chloride)

A

chelating agents

148
Q

ammonium form of polystyrene

A

ion exchange resins

149
Q

it uses formic acid-containing decalcifying solutions to increase solubility

A

ion exchange resins

150
Q

complete decalcification can be measured using physical or x-ray method

A

ion exchange resins

151
Q

it is not recommended for fluids that contain mineral acids (nitric acid, HCl)

A

ion exchange resins

152
Q

attraction of positively charged calcium ions

A

electrophoresis

153
Q

makes use of 88% formic acid; requires shorter time to remove calcium and is suitable for small bone fragments

A

electrophoresis

154
Q

tests to measure extent of decalcification:

A
  1. physical/mechanical
  2. x-ray/radiologic
  3. chemical/calcium oxalate
  4. bubble test
155
Q

bending of tissue or probing using a needle; prone to produce artifacts and destroys cellular details

A

physical/mechanical

156
Q

most reliable, ideal, and sensitive, but it is very expensive; not for mercuric chloride fixatives due to radiopacity

A

x-ray/radiologic

157
Q

by calcium hydroxide/calcium oxalate precipitation;
(+) precipitation = incomplete decalcification

A

chemical/calcium oxalate

158
Q

uses calcium carbonate;
(+) bubbles = incomplete decalcification

A

bubble test

159
Q

tissue softeners:

for hard tissues to facilitate cutting and processing

A
  1. lendrum’s (4% phenol)
  2. perenyi’s fluid
  3. molliflex (tissue may appear swollen and soapy)
  4. 2% HCl
  5. 1% HCl in 70% alcohol
160
Q

post decalcification:

to remove/neutralize acid from tissues

A
  1. lithium carbonate
  2. 5-10% sodium carbonate
  3. rinsing in running tap water
  4. formol saline with 15% sucrose
  5. phosphate buffered saline with 15-20% sucrose
161
Q

to remove fixative and water; done through ascending grades of alcohol

A

dehydration

162
Q

ratio of dehydrating agent to tissue

A

10:1

163
Q

initial concentration of alcohol for routine tissues

A

70%

164
Q

initial concentration of alcohol for delicate tissues

A

30%

165
Q

types of dehydrating agents:

A
  1. alcohols
  2. acetone
  3. dioxane
  4. cellosolve
  5. tri-ethyl phosphate
  6. tetrahydrofuran
166
Q

other dehydrating agents for EM

A
  1. ethanol
  2. propylene oxide
  3. acetonitrile
167
Q

routinely used for dehydration

A

alcohols

168
Q

<70% alcohol may _____ tissues

A

macerate

169
Q

dehydration hastens at _____

A

37C

170
Q

it ensures complete dehydration; it removes water from dehydrating agent;
(+) blue = presence of water

A

anhydrous copper sulfate

171
Q

most common (routine) and best dehydrating agent; fast-acting, penetrates tissues easily, and not toxic

A

ethanol

172
Q

for blood and tissue films, and blood smears; can lead to blindness and death

A

methanol

173
Q

for plant and animal microtechniques; slow-acting, but less shrinkage

A

butanol

174
Q

a universal solvent (dehydrating and clearing agent)

A

tertiary butanol
dioxane
tetrahydrofuran

175
Q

alcohol that dissolves paraffin

A

pentanol

176
Q

excellent ethanol substitute; best clearing agent for microwave techniques

A

isopropanol

177
Q

mixture of ethanol + methanol

A

denatured alcohol

178
Q

highly miscible but flammable

A

acetone

179
Q

fast-acting; for most urgent biopsies; it can cause considerable shrinkage and brittleness; lipids are removed

A

acetone

180
Q

diethylene oxide

A

dioxane

181
Q

miscible with paraffin, alcohol, xylene; produces less shrinkage and tissues can be stored for longer period; expensive, tissues tend to ribbon poorly and highly toxic

A

dioxane

182
Q

uses pure dioxane and paraffin

A

Graupner’s

183
Q

uses dioxane and anhydrous calcium oxide/quicklime

A

Weiseberger’s

184
Q

ethylene glycol monoethyl ether

A

cellosolve

185
Q

rapid, and tissues can be stored for longer periods w/o distortion; combustible at 110-120F, toxic, and decompose on sunlight

A

cellosolve

186
Q

for dehydrating sections and smears; removes water very easily, and produces minimum shrinkage and distortion

A

tri-ethyl phosphate

187
Q

dissolves many substances, including fats; can cause conjunctival irritation and odorous

A

THF

188
Q

transition fluid; for resin infiltration

A

propylene oxide

189
Q

propylene oxide substitute

A

acetonitrile

190
Q

to remove the dehydrating agent; “dealcoholization”

A

clearing

191
Q

miscible with dehydrating agent and embedding medium; makes the tissues translucent/transparent; flammable liquids with low boiling point

A

clearing agent

192
Q

types of common clearing agents:

A
  1. xylene (xylol)
  2. toluene (toluol)
  3. benzene
  4. chloroform
  5. cedarwood oil
193
Q

other clearing agents:

A
  1. aniline oil
  2. clove oil
  3. carbon tetrachloride
  4. oils of bergamot, origanum, wintergreen
  5. carbon disulfide & phenol
  6. terpineol
  7. limonene
  8. high test aviation lead free gasoline
194
Q

clearing agents for double-embedding:

A
  1. methyl benzoate
  2. methyl salicylate
  3. amyl acetate
195
Q

routine and most rapid clearing agent

A

xylene

196
Q

Xylene

*clearing time: _____
*refractive index: _____
*for tissue sections with _____
*for _____ sections, but not for _____ and _____ tissues
*turns _____ when tissues are incompletely dehydrated

A

15-20 mins to 1 hour

1.48 to 1.50

<5mm thick

celloidin; nervous and lymphatic

milky

197
Q

xylene and benzene substitute

A

toluene

198
Q

tissues do not become excessively hard or brittle even if left for 1 day; not carcinogenic, but toxic upon prolonged exposure

A

toluene

199
Q

can cause aplastic anemia

A

benzene

200
Q

for urgent biopsies (15-60 mins); causes minimum shrinkage to tissues

A

benzene

201
Q

clearing agent for tough tissues

A

chloroform

202
Q

slower than xylene and hepatotoxic, but causes less brittleness

A

chloroform

203
Q

for tissue blocks up to 1 cm, and can be used for vacuum embedding

A

chloroform

204
Q

tissues do not become translucent and tend to float

A

chloroform

205
Q

clearing agent for CNS, smooth muscles, and skin

A

cedarwood oil

206
Q

it requires two changes to properly be used, and difficult to remove

A

cedarwood oil

207
Q

no tissue distortion, but very slow; turns milky upon prolonged storage

A

cedarwood oil

208
Q

clearing agent for delicate tissues, embryos and insects

A

aniline oil

209
Q

tissues tend to become aduterated

A

clove oil

210
Q

everything similar to chloroform, but cheaper

A

carbon tetrachloride

211
Q

oils of _____ (skin), _____ (skin and smooth muscle, _____

A

bergamot, origanum, wintergreen

212
Q

clearing agent for smooth muscles

A

carbon disulfide and phenol

213
Q

clearing agent for eyes

A

terpineol

214
Q

obtained from citrus fruits and with a faint pleasant odor

A

limonene

215
Q

it replaces the clearing agent with an infiltrating medium to fill all cavities

A

infiltration (impregnation)

216
Q

ratio of infiltrating medium to tissue

A

25:1

217
Q

types of infiltrating media:

A
  1. paraffin
  2. celloidin
  3. gelatin
  4. plastic (resin)
218
Q

simplest, most common and best infiltrating medium

A

paraffin

219
Q

introduced by Butschlii; not recommended for fatty tissues

A

paraffin

220
Q

duration of paraffin infiltration

A

<24 hours

221
Q

melting point of paraffin

A

56 C

222
Q

low MP: _____
high MP: _____

A

paraffin is soft
paraffin is hard

223
Q

overheated paraffin temperature: tissues become brittle, with shrinkage and hardening

A

60 C

224
Q

filtered in wax oven using Green’s 904/coarse filter

A

fresh wax

225
Q

used twice only; water removed by heating wax at 100-105 C

A

reused wax

226
Q

for extra hardness, paraffin can be mixed with _____

A

10-20% beeswax or ceresin

227
Q

methods of paraffin wax impregnation:

A
  1. manual
  2. automatic
  3. vacuum
228
Q

requires 4 changes of wax at 15-minute intervals; temperature: 2-5 C above MP

A

manual

229
Q

use of automatic tissue processor; with 2 thermostatically controlled wax baths

A

automatic

230
Q

wax bath thermostat

A

3 C above MP

231
Q

done inside an embedding oven (2-4 C above MP)

A

vacuum

232
Q

impregnation under negative atmospheric pressure; fastest method (time reduced from 25-75% of normal time) and hastens the removal of air bubbles

A

vacuum

233
Q

paraffin wax substitutes:

A
  1. paraplast
  2. embeddol
  3. ester wax
  4. bioloid
  5. tissue mat
  6. water-soluble wax
234
Q

paraffin + synthetic plastic polymer (DMSO)
MP: 56-57 C

A

paraplast

235
Q

similar to paraplast, but MP: 56-58 C

A

embeddol

236
Q

prior clearing is not required; insoluble to water, but soluble to 95% ethanol

A

ester wax

237
Q

harder than paraffin (use sliding/sledge type microtome)
MP: 46-48 C

A

ester wax

238
Q

semisynthetic embedding for eyes

A

bioloid

239
Q

embedding medium made of rubber

A

tissue mat

240
Q

MP is 38-42 C or 45-56 C

A

polyethylene glycols

241
Q

most common example of water-soluble wax

A

carbowax

242
Q

prior dehydration and clearing is required; used for enzyme histochemistry

A

water-soluble wax

243
Q

hygroscopic (dissolves when fishing out)

A

water-soluble wax

244
Q

purified form of nitrocellulose

A

celloidin

245
Q

embedding medium used for specimen with large, hollow cavities; dissolve in equal parts of ether and alcohol; slow but does not require heat during processing

A

celloidin

246
Q

methods of celloidin impregnation:

A
  1. wet method
  2. dry method
  3. nitrocellulose
247
Q

celloidin technique that uses equal parts of ether and 70% alcohol; for bones, brain, teeth

A

wet method

248
Q

celloidin technique that uses Gilson’s mixture (chloroform & cedarwood oil); for eyes

A

dry method

249
Q

preferred since it produces harder blocks & thinner sections; soluble in equal parts of ether and alcohol

A

low viscosity nitrocellulose

250
Q

to prevent crackling of LVN, add _____

A

plasticizer (castor oil)

251
Q

embedding medium that is rarely used; for frozen sections, histochemical and enzyme studies

A

gelatin

252
Q

embedding medium that is water soluble (prior dehydration and clearing is not required); tissues should be <2-3 mm thick

A

gelatin

253
Q

to prevent molds, add _____

A

1% phenol

254
Q

aka. resin; for EM (80nm thick), high resolution light microscopy (renal biopsies and hematopoietic tissues) and extremely hard tissues

A

plastic

255
Q

plastic classifications according to chemical composition:

A
  1. epoxy
  2. polyester
  3. acrylic
256
Q

resin with widest application; for LM and choice for TEM; has the ability to produce sections with thickness of 30-40 nm

A

epoxy

257
Q

epoxy resins:

A
  1. bisphenol A
  2. glycerol
  3. cyclohexane dioxide (spurr)
258
Q

most stable epoxy resin

A

bisphenol A

259
Q

fastest epoxy resin, but highly toxic

A

cyclohexane dioxide

260
Q

resin that is seldom used; for EM

A

polyester

261
Q

resin preferred for high resolution LM

A

acrylic

262
Q

acrylic for uncalcified bones

A

acrylates (methyl methacrylates)

263
Q

acrylic for TEM

A

methacrylates (polyglycol/glycol methacrylates)

264
Q

it is done after wax impregnation

A

embedding (casting/blocking)

265
Q

orientation: surface of section should be _____ to the bottom of mold

A

parallel

266
Q

temperature in embedding

A

5-10 (6-10) C above MP of wax

267
Q

molds used:

A
  1. leukhart’s mold
  2. compound unit
  3. plastic rings and base molds
  4. disposable molds
  5. watch glass
  6. TIMS
268
Q

2 L-shaped strops of heavy brass or metal; size of mold is adjustable

A

leukhart’s mold

269
Q

made of interlocking grids or compartments

A

compound unit

270
Q

stainless steel base mold fitted with a plastic embedding ring

A

plastic rings and base molds

271
Q

disposable molds:

A
  1. peel away - even block w/o trimming
  2. ice tray - from ordinary refrigerators
  3. paper boat - cheap and easy to make
272
Q

molds used for fragmentary biopsies

A

watch glass

273
Q

1st infiltration w/ celloidin, 2nd infiltration w/ paraffin

A

double embedding

274
Q

done hand in hand w/ embedding if individual mode is used due to solidification that produces blocks

A

blocking

275
Q

process of removing excess wax to allow easy sectioning

A

trimming

276
Q

formation after trimming

A

truncated pyramid

277
Q

at least _____ of wax should surround the block

A

2mm

278
Q

process where cold, embedded tissue is cut into uniformly thin pieces via microtome

A

sectioning

279
Q

microtome is invented by

A

Wilhelm His Sr.

280
Q

it is used for unfixed, unfrozen tissue and uses a vibrating blade

A

vibratome

281
Q

essential parts of microtome

A
  1. block holder (chuck)
  2. knife carrier with knife
  3. pawl, ratched feed wheel, and adjustment screws
282
Q

types of microtome:

A
  1. rocking/cambridge
  2. rotary
  3. sliding
  4. freezing
  5. ultrathin
283
Q

simplest microtome used for large paraffin tissues, but not serial sections

A

rocking/cambridge

284
Q

invented by Trefall

A

rocking/cambridge

285
Q

routinely used microtome for paraffin and serial sections

A

rotary

286
Q

invented by Minot

A

rotary

287
Q

microtome used for celloidin and hard, rough tissue blocks

A

sliding

288
Q

invented by Adams, further developed by Alexander Cummings

A

sliding

289
Q

preferred type of sliding microtome; knife is held rigidly in clamps

A

base sledge

290
Q

dangerous type of sliding microtome; knife is moving

A

standard

291
Q

microtome for frozen section; with a propellant (CO2)

A

freezing

292
Q

invented by Queckett

A

freezing

293
Q

also known as cold microtome

A

cryostat

294
Q

temperature of cryostat

A

-5 to 30 C (ave 20 C)

295
Q

microtome used for electron microscopy; tissues are 0.5 um thick embedded in resin

A

ultrathin

296
Q

knife used in ultrathin microtome

A

diamond knife

297
Q

types of microtome knives:

A
  1. plane concave
  2. biconcave
  3. plane wedge
  4. disposable knives
  5. glass (diamond knives)
298
Q

knife used for rocking and rotary microtome

A

plane concave

299
Q

plane concave knife size

A

25 mm

300
Q

less concave side is for

A

celloidin

301
Q

more concave side is for

A

paraffin

302
Q

knife used for rotary microtome, paraffin sections; both sides are concave

A

biconcave

303
Q

biconcave knife size

A

120 mm

304
Q

knife used for sliding microtome, and for very hard tissues and frozen sections

A

plane wedge

305
Q

plane wedge knife size

A

100 mm

306
Q

knife coated with polytetrafluoroethylene for easy ribboning

A

disposable knives

307
Q

has an angle of 27-32; cutting facet; on the tapered edge of knife

A

bevel angle

308
Q

has an angle of 15; with maximum tissue penetration and less distortion

A

cutting angle

309
Q

has an angle of 0-15; between the block surface and cutting edge of knife

A

clearance angle

310
Q

angle between upper surface of facet and surface of block

A

rake angle

311
Q

angle between sides of knife

A

wedge angle

312
Q

done for sharpening and removing nicks; from heel to toe by 10-20 strokes

A

honing

313
Q

types of hones:

A
  1. belgian yellow (best result)
  2. arkansas
  3. fine carborundum (badly nicked)
  4. plate glass (stone substitute)
314
Q

done for polishing and removing burrs; from toe to heel by 40-120 double strokes

A

stropping

315
Q

it is made of horse leather
(3-4 x 18 inch); should be oiled in the back (do not use mineral oil)

A

paddle strops

316
Q

types of sections produced:

*paraffin
*celloidin
*renal biopsy
*ultrathin
*frozen

A

4-6 um
10-15 um
2 um (plastic embedding medium)
80 nm (silver/straw colored)
4 um (rotary microtome)

317
Q

done to flatten tissues after sectioning

A

water bath

318
Q

done to obtain sections from water bath to the slide

A

fishing out

319
Q

duration of tissues in water bath

A

30 secs to 1-2 mins

320
Q

drying the slide can be done by:

A
  • drain at an angle of 60-85 for 2-5 mins
    *hot plate at 45-55C for 30-45 mins
  • overnight incubator: best for nervous tissues
    *wax oven at 56-60C for 20-30 mins
    *urgent method: above a bunsen burner until the wax melt
321
Q

adhesives

A
  1. Mayer’s Egg Albumin
  2. 1% gelatin
  3. cellulose
  4. poly L-lysine
  5. sodium silicate
  6. resins
322
Q

most commonly used adhesive; not recommended for exfoliative cytology

A

Mayer’s egg albumin

323
Q

components of mayer’s egg albumin

A
  1. egg white
  2. glycerol: increases viscosity and prevent drying
  3. thymol: prevent mold growth
324
Q

with firmer attachment than albumin; gently heated before use to melt; disadvantage: stains with many dyes

A

1% gelatin

325
Q

general purpose adhesive for immunohistochemistry

A

poly L-lysine

326
Q

a commercial syrup with 1:10 dilution; not affected by mild alkaline solutions

A

sodium silicate

327
Q

adhesive disadvantages to staining:

  1. blackening in silver impregnation and reticulin
  2. reddening in methyl green-pyronin
A

sodium silicate

328
Q

with great adhesion

A

resins

329
Q

made of epoxy resins diluted with 1:10 acetone

A

araldite

330
Q
A

MTLE (3 decks)

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