LESSON 2: FIXATION Flashcards
1
Q
first and most critical step in tissue processing
A
FIXATION
2
Q
fixing or preserving fresh tissue for examination
A
FIXATION
3
Q
should be done immediately to preserve cellular and tissue morphology
A
FIXATION
4
Q
Two Purposes of Fixation
A
- Preservation: primary purpose
- Protection: secondary purpose
5
Q
capable of forming cross-links between proteins
A
Fixative agents
6
Q
stabilizes tissue components making them insoluble to lysosomal enzymes
A
Fixative agents
7
Q
Types of Fixative:
A
- Additive: becomes part of the cross-link itself
- Non-Additive: facilitates the removal of water in order for cross-links to form
8
Q
: becomes part of the cross-link itself
A
- Additive
9
Q
: facilitates the removal of water in order for cross-links to form
A
- Non-Additive
10
Q
capable of inactivating lysosomes
A
Fixative agents
11
Q
RETARDED BY:
A
Increase in size and thickness
Presence of mucus
Presence of fats
Presence of blood
Decrease in temperature
12
Q
ENHANCED BY:
A
Decrease in size and thickness
Presence of agitation
Presence of heat
13
Q
pH:
A
6 to 8
14
Q
Routine Manual:
A
Room Temp (20 to 22oC)
15
Q
Routine Automated:
A
40 oC
16
Q
Electron Microscopy:
A
0 to 4oC
17
Q
Formalin at 60 oC:
A
very urgent biopsies
18
Q
Light Microscopy:
A
2cm2 by 0.4cm thick
19
Q
Electron Microscopy:
A
1 to 2 mm2
20
Q
Lung Edema:
A
1 to 2 cm thick
21
Q
Light microscopy:
A
slightly hypertonic (400-450 mOsm)
22
Q
Electron Microscopy:
A
more or less isotonic (340 mOsm)
23
Q
Formaldehyde:
Glutaraldehyde:
A
10%
3%
24
Q
Routine:
A
10 to 25 times the volume of the specimen
25
Museum:
50 to 100 times the volume of the specimen
26
Cover with several layers of gauze
1. Air-filled Lungs
27
Dilate with cotton soaked in fixative
2. Hollow Organs
28
Completely open specimen
2. Hollow Organs
29
Fix first before sampling
3. Brain
30
Suspended by a cord tied under the Circle of Willis
3. Brain
31
Intravascular perfusion using Ringer’s lactate
3. Brain
32
Fixed whole
4. Eyes
33
Inject formol-alcohol
4. Eyes
34
Lendrum’s Method: washed out with running water overnight and immersed in 4% aq. phenol solution for 1-3 days
5. Hard Tissues
35
Stretched with sutures on each end
6. Muscles
36
Laid flat in a moist filter paper
6. Muscles
37
CHARACTERISTICS OF A GOOD FIXATIVE
1. Cheap & economical
2. Stable and safe to handle
3. Fast acting, permits rapid an even penetration
4. Inhibits bacterial decomposition & autolysis
5. Must harden tissues
6. At least isotonic
7. Must render tissues insensitive to subsequent processing
8. Must be compatible with many staining procedures
38
: made up of only one component substance
1. Simple Fixatives
39
: made up of two or more fixatives
2. Compound Fixatives
40
: permit general microscopic study of tissue structures and normal intercellular relationship of tissues
1. Microanatomical Fixatives
41
: preserve specific cellular components
2. Cytological Fixatives
42
: preserve nuclear structures
A. Nuclear Fixatives
43
contain glacial HAc
A. Nuclear Fixatives
44
high affinity to nuclear chromatin
glacial HAc
45
destroys mitochondria and Golgi bodies
glacial HAc
46
pH 4.6 or less
A. Nuclear Fixatives
47
: preserve cytoplasmic structures
Cytoplasmic Fixatives
48
do not contain glacial HAc
Cytoplasmic Fixatives
49
pH of more than 4.6
Cytoplasmic Fixatives
50
: preserve chemical constituents
Histochemical Fixatives
51
a. Lipids:
mercuric chloride or potassium dichromate
52
b. Phospholipids:
Baker’s formol-calcium
53
c. Cholesterol:
digitonin
54
d. Carbohydrates:
alcoholic fixatives
55
e. Glycogen:
Rossman’s fluid or cold absolute alcohol
56
f. Proteins:
neutral buffered formol saline or formaldehyde vapor
57
g. Electron microscopy:
double fixation
58
1. Electron Cytochemistry:
Karnovsky’s paraformaldehydeglutaraldehyde solution
59
2. Acrolein:
mixture of glutaraldehyde or fomaldehyde
60
rapid penetration, preserves morphology and enzyme activity at low concentrations
Acrolein
61
immersion fixation of surgical biopsies
Acrolein
62
: for routine paraffin sections, electron microscopy, histochemistry and enzyme studies
ALDEHYDE FIXATIVES
63
gas produced by the oxidation of methyl alcohol
Formaldehyde (Formalin)
64
buffered at ph 7 to 8
Formaldehyde (Formalin)
65
hypoxia in tissues leads to acidity which favors the formation of Formalin heme pigments (black, polarizable deposits)
Formaldehyde (Formalin)
66
Formaldehyde (Formalin) Pure Stock:
40% formalin
67
Formaldehyde (Formalin) Dilution:
1:10 (10% solution); 1:20 (5% solution)
68
Formaldehyde (Formalin) Concentration for fixation:
10% formalin
69
: white precipitate due to prolonged storage
Paraformaldehyde
70
may be removed by filtration or addition of 10% methanol
Paraformaldehyde
71
central nervous tissues and general post-mortem tissues for histochemical examination
10% Formol-Saline
72
ideal with most stains including silver impregnation
10% Formol-Saline
73
duration of fixation: more than 24 hours (slow fixative)
10% Formol-Saline
74
preservation and storage of surgical, post-mortem and research specimens
10% Neutral Buffered Formalin
75
best fixative for frozen sections
10% Neutral Buffered Formalin
76
best fixative for iron pigments and elastic fibers
10% Neutral Buffered Formalin
77
routine post-mortem tissues
Formol-Corrosive
78
for lipids, especially neutral fats and phospholipids
Formol-Corrosive
79
no need for washing-out
Formol-Corrosive
80
used to fix sputum
Alcoholic Formalin
81
for the demonstration of immunoperoxidase activity
Alcoholic Formalin
82
made up of two formaldehyde residues linked by three carbon chains
Glutaraldehyde
83
used in conjunction with osmium tetroxide
Glutaraldehyde
84
Glutaraldehyde Fixation time:
½ hour to 2 hours
85
most common metallic fixative
Mercuric Chloride
86
tissue photography
Mercuric Chloride
87
tissues contain black precipitates of mercury (except Susa)
Mercuric Chloride
88
fixing small pieces of liver, spleen, connective tissues fibers and nuclei
Zenker’s Fluid
89
: removal of mercuric deposits in tissues
De-zenkerization
90
pituitary gland, bone marrow and blood containing organs (spleen and liver)
B. Zenker-formol
91
for tumor biopsies
C. Heidenhain’s Susa Solution
92
for bone marrow biopsies
D. B-5 Fixative
93
preserves carbohydrates
A. Chromic Acid
94
preserves lipids and mitochondria
B. Potassium Dichromate
95
demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissues
C. Regaud’s Fluid
96
study of early degenerative processes and tissue necrosis
D. Orth’s Fluid
97
demonstrates Rickettsia and other bacteria
D. Orth’s Fluid
98
demonstration of acid mucopolysaccharides
Lead Fixatives
99
are soluble in water, therefore, tissues should not be washed in running tap water (use 70% alcohol instead)
Picrates
100
fixation of embryos and pituitary biopsies
1. Bouin’s Solution
101
fixative for glycogen
2. Brasil’s Alcoholic Picroformol Fixative
102
fixation of nucleoproteins
GLACIAL ACETIC ACID
103
Preserves but does not “fix” glycogen granules
1. 95 % Ethanol
104
For dry and wet smears, blood and bone marrow samples
2. Methanol
105
for touch preparations
3. Isopropyl alcohol (95%)
106
most rapid fixative
4. Carnoy’s fluid
107
for chromosomes, lymph glands, urgent biopsies and brain tissue for the diagnosis of rabies
4. Carnoy’s fluid
108
demonstration of mucopolysaccharides and nucleoproteins
5. Newcomer’s
109
Permanently fixes fats and recommended for fixing nuclear structures
1. Flemming’s (chrome-osmium HAc)
110
For cytoplasmic structures
2. Flemming’s w/o acetic acid
111
may also be used as a weak decalcifying agent
TRICHLOROACETIC ACID
112
use at cold temperature (-5 to 4oC)
ACETONE
113
fixation of brain tissues for rabies diagnosis
ACETONE
114
Routinely used fixative
Formaldehyde
115
Used for electron microscopy
Glutaraldehyde
116
For enzymes, nucleoproteins, fats and mucins
10% Formol-Saline
117
Best for frozen sections, iron pigments and elastic fibers
10% Neutral Buffered Formalin
118
For lipids, especially neutral fats and phospholipids
Formol Corrosive/Sublimate
119
For immunoperoxidase activity, glycogen, microincineration & sputum
Gendre’s
120
For small pieces of liver, spleen, connective tissue fibers & nuclei
Zenker’s
121
For pituitary, bone marrow and blood-containing organs
Zenker-Formol
122
For tumor biopsies of the skin
Heidenhain’s SuSa
123
For bone marrow biopsies
B-5
124
For carbohydrates
Chromic Acid
125
For lipids and mitochondria
Potassium dichromate
126
For chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissues
Regaud’s
127
For early degenerative processes, tissue necrosis, Rickettsiae and other bacteria
Orth’s
128
For acid mucopolysaccharides
4% Basic Lead acetate
129
For embryos and pituitary biopsies
Bouin’s
130
For glycogen
Brasil’s Alcoholic Picroformol
95% Ethanol
131
For dry and wet smears, blood and bone marrow samples
100% Methanol
132
For touch preparations
95% Isopropyl alcohol
133
For chromosomes, lymph glands, urgent biopsies and brain fixation for rabies diagnosis
Carnoy’s
134
For mucopolysaccharides and nuclear proteins
Newcomer’s
135
For fats and nuclear structures
Flemming’s with HAc
136
For cytoplasmic structures
Flemming’s without HAc
137
For nuclear structures
Glacial Acetic acid
138
For dense fibrous tissues
Trichloroacetic acid
139
For brain fixation for rabies diagnosis and water-diffusible enzymes
Acetone
140
placing a fixed tissue in a second fixative
POST MORDANTING/SECONDARY FIXATION
141
2.5-3% potassium dichromate as the secondary fixative
Post-chromatization
142
acts as a mordant for better staining effects
2.5-3% potassium dichromate
143
1. Tap Water removes excess
C
F
O
144
1. Tap Water:
Chromates (Helly’s, Zenker’s & Flemming’s)
Formalin
Osmic Acid
145
2. 50 – 70 % Alcohol:
Picric acid (Bouin’s solution)
146
3. Alcoholic iodide:
Mercuric fixatives
147
: preserve cellular structure & require short fixation time
FIXATIVES
148
ALDEHYDE FIXATION
1. Karnovsky’s paraformaldehyde - glutaraldehyde
2. Acrolein – glutaradehyde / formaldehyde
149
POST FIXATION:
Buffered Osmium Tetroxide
150
OSMIC ACID FIXATION
1. Palade’s fixative
2. Zetterqvist Osmium fixative
3. Millonig’s Osmium tetroxide
151
Enzyme Histochemistry
1. [?]
2. Frozen sections: [?]
4% formaldehyde or formol-saline
acetone or formaldehyde
152
Fixatives for Smears
1. Schaudinn’s
Mercuric Chloride Absolute Ethanol Acetic acid
2. Ether-Alcohol
3. Methanol
153
Other Methods of Fixation
1. Heat
2. Microwave Fixation
154
Microwave Fixation –
vacuum, heat & agitation (10 – 15 mm) + formaldehyde fixatives
155
Chemical fixation is avoided
SPECIAL TECHNIQUES
156
Recommended for histochemical studies
SPECIAL TECHNIQUES
157
Rapid freezing of tissue blocks to allow instant cessation of cellular activities
Quenching
158
Freezing Agents
1. Liquid Nitrogen
2. Isopentane
3. Pentane
4. Propane
5. Dichlorodifluoromethane
159
: tissue plunged in isopentane or propane-isopentane (-160 to -180 OC)
Rapid freezing
160
: removal of tiny ice crystals by sublimation in a vacuum (-30 to -40 OC)
Desiccation
161
Similar with freeze-drying
FREEZE-SUBSTITUTION
162
FREEZE-SUBSTITUTION
Instead of dehydration in a vacuum, tissue blocks are:
Fixed in Rossman’s fluid or 1% acetone
Dehydrated with abs. alcohol
