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Fixation Flashcards

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1
Q

Primary & Secondary Aim of fixation

A

1° - Preserve
2° - Protect & harden tissue

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2
Q

Recommended volume (fixative:tissue)

A

10-20:1

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3
Q

Volume ratio for
1. osmium tetraoxide
2. museum prep
3. traditional

A
  1. 5-10:1
  2. 50-100:1
  3. 10-25:1
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4
Q

pH for fixative

A

Neutral 7 (6-8 pH)

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5
Q

thickness of the section

A

LM: 2 cm2
EM: 1-2 mm2

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6
Q

osmolality of fixative

A

400-450 mOsm/kg (slightly hypertonic)

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7
Q

Fixative must be ___ causing minimal alterations of cells & constituents

A

isotonic

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8
Q

Physical methods of fixation

A
  • Heat
  • Microwave
  • Freeze drying
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9
Q

Metallic fixative
Examples of Mercuric chloride (BHZZ)

A

B-5 fixative
Heidenhain’s susa
Zenker’s
Zenker-formol (Hellu’s)

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10
Q

Metallic fixative
Examples of chromate (CROP)

A

Chromic acid
Regaud’s soln
Orth’s soln
Potassium dichromate

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11
Q

Picric acid fixatives

A

Bouin’s
Brasil’s
“BB Picric”

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12
Q

Alcohol fixatives

A

“MINCE”
Methyl alcohol (100%)
Isopropyl alcohol (95%)
Newcomer’s
Carnoy’s
Ethyl alcohol (70-100%)

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13
Q

Osmium tetraoxide fixative

A

Flemming’s soln w/ or w/o acetic acid

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14
Q

Micro anatomical fixatives must never contain

A

Alcohol & osmium tetraoxide (inhibits hematoxylin)

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15
Q

Nuclear fixatives contains __ at a pH of __

A

glacial acetic acid
pH of 4.6 or less

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16
Q

Cytoplasmic fixatives must never contain

A

“OFF HeR”
glacial acetic acid (destroys mitochondria & golgi bodies)
pH of more than 4.6

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17
Q

Histochemical fixative includes

A

“10% FANA”
10% formalin
Abs ethanol
Newcomer’s
Acetone

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18
Q

Both microanatomic & nuclear fixative

A

Bouin’s & Heindenhain’s susa

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19
Q

Both histochemical & nuclear

A

Newcomer’s

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20
Q

Both microanatomical & histochemical

A

10% formol saline

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21
Q

Both microanatomic & cytoplasmic

A

Helly’s (Zenker formol)

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22
Q

Factors that enhance fixation

A
  • Size & thickness (⬇️)
  • Agitation
  • Moderate heat
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23
Q

Factors that retard fixation

A

Increase size & thickness
Cold temp
Presence of MBF (mucus, blood, fat)

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24
Q

Creating a covalent bond between protein & aldehyde

A

Cross-linking fixative

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25
Act by reducing the solubility of protein molecules & disrupting hydrophobic interactions
Precipitating/denaturing
26
Done before dehydration & before staining
Secondary fixation
27
form of 2° fixation where tissue is placed in 2.5-3% potassium dichromate for 24 hrs for cyto preservation
post-chromatiziation
28
Process of removing fixative
Washing out
29
Used to remove excessive mercuric fixative
Alcohol iodine
30
Used to remove excess picric acid
50-70% alcohol
31
Fixatives effective for preservation of lipids
mercuric chloride & potassium dichromate & formalin X alcohol & acetone
32
Fixatives for carbohydrates
alcoholic fixatives
33
Fixatives for amino acid histochemistry
NBF saline or Formaldehyde vapor
34
Fixatives for electron microscopy
"GOPKZ" Glutaraldehyde Osmium tetraoxide Paraformaldehyde Karnovsky's Zamboni
35
Satisfsctory fixative for both morpho & IHC
Formalin
36
artifact eliminated or reduced by fixation in phenol-formalin how to completely stop the formation?
Formalin pigments using NBF
37
Artifacts found in liver biopsies with an intense eosinophilic staining (in H&E stains)
Crush artifacts
38
Difficulty: Removal of substances soluble in fixing agent Cause?
Wrong choice of fixative
39
Difficulty: Presence of artifact pigments Cause?
Incomplete washing of fixative
40
Difficulty: Tissues are soft & feather-like consistency Cause?
Incomplete fixation
41
Difficulty: Shrinkage & swelling of cells & tissue structure Cause?
Overfixation
42
Difficulty: Tissue blocks are brittle & hard Cause?
Prolonged fixation
43
What will happen if Formalin is unbuffered & used on blood containing tissues
Formation of abundant brown pigment granules (blackening of hemoglobin)
44
Good for - preserving fat & mucin - frozen tissues are easily prepared - colored tissue photography - nervous tissue demonstration
10% Formalin
45
Prolonged storage of formaldehyde at low temp causes
Precipitation of white paraformaldehyde deposites
46
How to remove paraformaldehyde deposits
Filtration or adding 10% methanol
47
Concentrated solution should NEVER be __ since it might violently explode
Neutralized
48
Removal of formalin pigments
- Picric acid - Kardasewitsch's - Lillie's
49
Best fixative for iron pigments
10% Neutral Buffered Formalin
50
Composition for 10% NBF
Sodium dihydrogen phosphate Disodium hydrogen phosphate Distilled H2O 40% formaldehyde
51
Recommended for routine post-mortem tissues
Formol-corrosive (formol-sublimate)
52
Composition for alcoholic formalin (Gendre's fluid)
95% ethanol saturated w/ picric acid Formaldehyde Glacial acetic acid
53
Depolymerizes back to formalin when heated
Paraformaldehyde
54
Satisfactory for EM & preserves cellular structures better than formaldehyde
Glutaraldehyde
55
All mixtures comtaining HgCl contain black precipitates except __
Susa
56
Removal of mercury deposits are done by
Dezenkerization: 0.5% iodine solution in 70% ethanol for 5-10 mins rinsed w/ H2O & decolorized for 5 mins
57
Mecuric chloride stock soln
Mercuric chloride Potassium dichromate Sodium sulfate Distilled H2O
58
DONT'S for mercuric chloride fixatives
Don't use metallic forceps & caps It must not go through the drain
59
Good for Trichrome staining & Tissue photography
Mercury chloride
60
Recommended for small pieces of liver, spleen but remember to add Glacial acetic acid before use
Zenker's fluid
61
Excellent for Pituitary gland, Bone marrow, Blood organs but remember to add formaldehyde before use
Zenker formol (Helly's)
62
Recommended for skin tumor biopsies that doesn't produce any black ppt
Heidenhain's susa
63
Used in 3% aq. soln that preserves mitochondria at a pH of 4.5 to 5.2
Potassium dichromate
64
Used for demonstration of Rickettsia
Orth's fluid
65
Recommended for fixing Golgi bodies, Mitochondria, RBCs & Colloid
Regaud's (Muller's)
66
Recommended for Acid mucopolysaccharides
Lead fixatives
67
It dyes the tissue yellow but can be removed by tx w/ acid/lithium carbonate Excellent for glycogen demonstration
Picric acid
68
Not suitable for fixing kidney structures, lipid & mucus
Bouin's
69
what happens to glacial acetic acid at 17°C
it solidifies
70
Fixes & precipitates nucleoproteins
Glacial acetic acid
71
If lesser concentrated solutions of alcoholic fixatives are used, what happens to the cell
swell
72
Gives the most usable DNA fragments for PCR
70-100% ethyl alcohol
73
Recommended for Chromosomes, Urgent biopsy, Rabies, Lymph nodes & its the fastest
Carnoy's
74
Rapid freezing Quenching temp: Sublimation temp:
Quenching: -160 to -180°C Sublimation: -30 to -40°C
75
Freeze drying substitute
Rossman's formula or in 1% acetone
76
If fixation is not immediately possible, what to do?
Refrigerate DO NOT FREEZE
77
Hollow organs should be packed with
cotton soaked fixative/completely opened before its immersed
78
How to fix human brains
must be floating & suspended by a cord tied under the circle of willis to prevent flattening
79
Should eyes be dissected before they are fixed? What to do?
NOOO Inject formol-alcohol before immersing
80
Water should not be used for these tissues bc its soluble
Glycogen
81
Wash excess mucus using
NSS
82
What method is used to fix hard tissues
Lendrum's method - immersing it in 4% aq phenol soln for 1-3 days

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