CHAPTER 7 CHEMICAL FIXATIVES Flashcards
1
Q
(e.g., Aldehydes)
A
Crosslinking Fixatives
2
Q
act by creating covalent chemical bonds between proteins in tissue.
A
Crosslinking Fixatives
3
Q
This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue.
A
Crosslinking Fixatives
4
Q
(e.g., alcoholic fixatives)
A
Precipitating (or denaturing) fixatives
5
Q
act by reducing the solubility of protein molecules and (often) by disrupting the hydrophobic interactions that give many proteins their tertiary structure.
A
Precipitating (or denaturing) fixatives
6
Q
The precipitation and aggregation of proteins is a very different process from the crosslinking that occurs with the aldehyde fixatives.
A
Precipitating (or denaturing) fixatives
7
Q
A
formaldehyde
8
Q
most commonly used fixative in histology
A
formaldehyde
9
Q
fixes the tissues by forming cross-linkages in the proteins, particularly between lysine residues; good for immunohistochemical techniques
A
formaldehyde
10
Q
The standard solution is [?] neutral buffered formalin or approximately [?] formaldehyde in phosphate- buffered saline.
A
10%
3.7%-4.0%
11
Q
is a gas produced by the oxidation of methyl alcohol, and is soluble in water to the extent of 37-40% weight in volume.
A
Formaldehyde
12
Q
Pure stock solution of [?] is unsatisfactory for routine fixation since high concentrations of formaldehyde tend to over-harden the outer layer of the tissue and affect staining adversely.
A
40% formalin
13
Q
is made with formaldehyde but the percentage denotes a different formaldehyde concentration.
A
Formalin
14
Q
The most widely used fixative for routine histology is [?]
A
10% neutral buffered formalin (NBF, approximately 4% formaldehyde), buffered to pH 7 with phosphate buffer.
15
Q
This fixative can effectively prevent autolysis and provide excellent preservation of tissue and cellular morphology.
A
10% neutral buffered formalin (NBF, approximately 4% formaldehyde)
16
Q
It is considered the fixative of choice for many other procedures that require paraffin embedding, including immunohistochemistry and interphase Fluorescent In-Situ Hybridization (FISH).
A
10% neutral buffered formalin (NBF, approximately 4% formaldehyde)
17
Q
FORMULA:
40% formaldehyde: 100 ml
Distilled water: 900 ml
Sodium dihydrogen phosphate monohydrate: 4 gm
Disodium hydrogen phosphate anhydrous 6.5 gm
A
10% Formal-Saline
18
Q
The solution should have a pH of 6.8
A
10% Formal-Saline
19
Q
Fixation time: 12 – 24 hours
A
10% Formal-Saline
Alcoholic formalin
20
Q
- It penetrates and fixes tissues evenly.
A
10% Formal-Saline
21
Q
- It preserves microanatomic and cytologic details with minimum shrinkage and distortion.
A
10% Formal-Saline
22
Q
- Large specimens may be fixed for a long time provided that the solution is changed every three months.
A
10% Formal-Saline
23
Q
- It preserves enzymes and nucleoproteins.
A
10% Formal-Saline
24
Q
- It demonstrates fats and mucin.
A
10% Formal-Saline
25
6. It does not over-harden tissues, thereby facilitating dissection of the specimen.
10% Formal-Saline
26
7. It is ideal for most staining techniques, including silver
impregnation.
10% Formal-Saline
27
8. It allows natural tissue color to be restored upon immersion in 70% alcohol.
10% Formal-Saline
28
1. It is a slow fixative. The period of fixation is required to be 24 hours or longer.
10% Formal-Saline
29
fixed tissues tend to shrink during alcohol dehydration; this may be reduced by secondary fixation.
10% Formal-Saline
30
3. Metachromatic reaction of amyloid is reduced.
10% Formal-Saline
31
4. Acid dye stains less brightly than when fixed with mercuric chloride.
10% Formal-Saline
32
1. It is cheap, readily available, easy to prepare, and relatively stable, especially if stored in buffered solution.
Formalin
33
2. It is compatible with many stains, and therefore can be used with various staining techniques depending upon the need of the tissues.
Formalin
34
3. It does not over-harden tissues, even with prolonged periods of fixation, as long as solutions are regularly changed.
Formalin
35
4. It penetrates tissues well.
Formalin
36
5. It preserves fat and mucin, making them resistant to subsequent treatment with fat solvents, and allowing them to be stained for demonstration.
Formalin
37
6. It preserves glycogen.
Formalin
38
7. It preserves but does not precipitate proteins, thereby allowing tissue enzymes to be studied. It does not make tissues brittle, and is therefore recommended for nervous tissue preservation.
Formalin
39
8. It allows natural tissue colors to be restored after fixation by immersing formalin-fixed tissues in 70% alcohol for one hour, and is therefore recommended for colored tissue photography.
Formalin
40
9. It allows frozen tissue sections to be prepared easily.
Formalin
41
10. It does not require washing out, unless tissues have stayed in formalin for excessively long periods of time.
Formalin
42
1. Fumes are irritating to the nose and eyes and may cause sinusitis, allergic rhinitis, or excessive lacrimation.
Formalin
43
2. The solution is irritating to the skin and may cause allergic dermatitis on prolonged contact.
Formalin
44
3. It may produce considerable shrinkage of tissues.
Formalin
Carnoy’s Fixative
45
4. It is a soft fixative and does not harden some cytoplasmic structures adequately enough for paraffin embedding.
Formalin
46
Reduces both basophilic and eosinophilic staining of cells, thereby reducing the quality of routine cytologic staining.
Formalin
47
Acidity of formic acid may, however, be used to an advantage when applying the silver impregnation technique of staining.
Formalin
48
It forms abundant brown pigment granules on blood-containing tissues, e.g., spleen, due to blackening of hemoglobin.
Formalin
49
Prolonged fixation may produce:
a. Bleaching of the specimen and loss of natural tissue colors.
b. Dispersal of fat from the tissue into the fluid.
c. Dissolution or loss of glycogen, and urate crystals
Formalin
50
solutions were devised as alternatives to mercuric chloride
formulations.
Zinc formalin (unbuffered)
51
FORMULA: Sat. Aq. Mercuric chloride 90 ml. Formaldehyde 40% 10 ml.
Formol-Corrosive (Formol-Sublimate)
52
Fixation time: 3-24 hours
Formol-Corrosive (Formol-Sublimate)
53
is recommended for routine post-mortem tissues.
Formol-mercuric chloride solution
54
1. It penetrates small pieces of tissues rapidly.
Formol-Corrosive (Formol-Sublimate)
55
2. It produces minimum shrinkage and hardening.
Formol-Corrosive (Formol-Sublimate)
56
3. It is excellent for many staining procedures including silver reticulum methods.
Formol-Corrosive (Formol-Sublimate)
57
4. It brightens cytoplasmic and metachromatic stains better than with formalin alone.
Formol-Corrosive (Formol-Sublimate)
58
5. Cytological structures and blood cells are well preserved.
Formol-Corrosive (Formol-Sublimate)
59
6. It fixes lipids, especially neutral fats and phospholipids.
Formol-Corrosive (Formol-Sublimate)
60
1. Penetration is slow; hence, tissue sections should not be more than 1 cm thick.
Formol-Corrosive (Formol-Sublimate)
61
2. It forms mercuric chloride deposits.
Formol-Corrosive (Formol-Sublimate)
62
3. It does not allow frozen tissue sections to be made.
Formol-Corrosive (Formol-Sublimate)
63
4. It inhibits the determination of the extent of tissue decalcification.
Formol-Corrosive (Formol-Sublimate)
64
is a polymerized form of formaldehyde, usually obtained as a fine white powder, which depolymerizes back to formalin when heated.
Paraformaldehyde
65
It is suitable for paraffin embedding and sectioning, and also for immunocytochemical analysis.
Paraformaldehyde
66
fixed samples can also be stained for general histology but the degree of fixation is less vigorous than Bouin’s so the quality of the morphology obtained will be less.
Paraformaldehyde
67
This fixative allows for subsequent immuno-detection of certain antigens and should therefore be used when the objective is to study morphology and protein expression simultaneously.
Paraformaldehyde
68
Its effects are reversible by excess water and it avoids formalin pigmentation.
Paraformaldehyde
69
Other benefits include: Long term storage and good tissue penetration.
Paraformaldehyde
70
is a mixture of paraformaldehyde and glutaral-dehyde.
Karnovsky’s Fixative (4% Paraformaldehyde-1% Glutaraldehyde in 0.1M Phosphate Buffer)
71
It is suitable for use when preparing samples for light microscopy in resin embedding and sectioning, and for electron microscopy.
Karnovsky’s Fixative (4% Paraformaldehyde-1% Glutaraldehyde in 0.1M Phosphate Buffer)
72
This fixative should always be prepared fresh.
Karnovsky’s Fixative (4% Paraformaldehyde-1% Glutaraldehyde in 0.1M Phosphate Buffer)
73
1. It has a more stable effect on tissues, giving a firmer texture with better tissue sections, especially of central nervous tissues.
Glutaraldehyde
74
2. It preserves plasma proteins better.
Glutaraldehyde
75
3. It produces less tissue shrinkage.
Glutaraldehyde
76
4. It preserves cellular structures better; hence, is recommended for electron microscopy.
Glutaraldehyde
77
5. It is more pleasant and less irritating to the nose.
Glutaraldehyde
78
6. It does not cause dermatitis.
Glutaraldehyde
79
1. It is more expensive.
Glutaraldehyde
80
2. It is less stable.
Glutaraldehyde
81
3. It penetrates tissues more slowly.
Glutaraldehyde
82
4. It tends to make tissue (i.e. renal biopsy) more brittle.
Glutaraldehyde
83
5. It reduces PAS positivity of reactive mucin.
Glutaraldehyde
84
This may be prevented by immersing glutaraldehyde-fixed tissues in a mixture of
concentrated glacial acetic acid and aniline oil.
85
1. The specimen vial must be kept refrigerated during the fixation process.
Glutaraldehyde
86
2. Solution may be changed several times during fixation by swirling the vials to make sure that the specimen is in contact with fresh solution all the time.
Glutaraldehyde
87
1. It is excellent for fixing dry and wet smears, blood smears and bone marrow tissues.
Methyl Alcohol 100%
88
2. It fixes and dehydrates at the same time.
Methyl Alcohol 100%
89
1. Penetration is slow.
Methyl Alcohol 100%
90
2. If left in fixative for more than 48 hours, t issues may be over hardened and difficult to cut.
Methyl Alcohol 100%
91
- is used for fixing touch preparations , although some touch preparations are air dried and not fixed, for certain special staining procedures such as Wright-Giemsa.
Isopropyl Alcohol 95%
92
is used at concentrations of 70-100%. If the lower concentrations are used, the RBC's become hemolyzed and WBC's are inadequately preserved.
Ethyl Alcohol
93
It may be used as a simple fixative.
Ethyl Alcohol
94
It is, however, more frequently incorporated into compound fixatives for better results.
Ethyl Alcohol
95
Fixation Time: 18-24 hours
Ethyl Alcohol
96
1. It preserves but does not fix glycogen.
Ethyl Alcohol
97
2. It fixes blood, tissue films and smears.
Ethyl Alcohol
98
3. It preserves nucleoproteins and nucleic acids, hence, is used for histochemistry, especially for enzyme studies.
Ethyl Alcohol
Carnoy’s Fixative
99
4. It fixes tissue pigments fairly well.
Ethyl Alcohol
100
5. It is ideal for small tissue fragments.
Ethyl Alcohol
101
1. Hemosiderin preservation is less than in buffered formaldehyde.
Ethyl Alcohol
102
2. It is a strong reducing agent; hence, should not be mixed with chromic acid, potassium dichromate and osmium tetroxide which are strong oxidizing agents.
Ethyl Alcohol
103
3. Lower concentrations (70-80%) will cause RBC hemolysis and inadequately preserve leukocytes.
Ethyl Alcohol
104
4. It dissolves fats and lipids, as a general rule. Alcohol-containing fixatives are contraindicated when lipids are to be studied.
Ethyl Alcohol
105
5. It causes glycogen granules to move towards the poles or ends of the cells (polarization).
Ethyl Alcohol
106
6. Tissue left in alcohol too long will shrink, making it difficult or impossible to cut.
Ethyl Alcohol
107
7. It causes polarization of glycogen granules.
Ethyl Alcohol
108
8. It produces considerable hardening and shrinkage of tissues.
Ethyl Alcohol
109
FORMULA:
Absolute alcohol 60 ml.
Chloroform 30 ml.
Glacial acetic acid 10 ml.
Carnoy’s Fixative
110
Fixation Time: 1-3 hours
Carnoy’s Fixative
111
It is considered to be the most rapid fixative and may be used for urgent biopsy specimens for paraffin processing within 5 hours.
Carnoy’s Fixative
112
It fixes and dehydrates at the same time.
Carnoy’s Fixative
Methyl Alcohol 100%
113
3. It permits good nuclear staining and differentiation.
Carnoy’s Fixative
114
4. It preserves Nissl granules and cytoplasmic granules well.
Carnoy’s Fixative
115
5. It preserves nucleoproteins and nucleic acids.
Carnoy’s Fixative
Methyl Alcohol 100%
116
6. It is an excellent fixative for glycogen since aqueous solutions are avoided.
Carnoy’s Fixative
117
7. It is very suitable for small tissue fragments such as curettings and biopsy materials.
Carnoy’s Fixative
Ideal: Ethyl Alcohol
118
8. Following fixation for one hour, tissues may be transferred directly to absolute alcohol-chloroform mixture, thereby shortening processing time.
Carnoy’s Fixative
119
9. It is also used to fix brain tissue for the diagnosis of rabies.
Carnoy’s Fixative
120
1. It produces RBC hemolysis, dissolves lipids and can produce excessive hardening and shrinkage.
Carnoy’s Fixative; Isopropyl Alcohol 95%
considerable: Ethyl Alcohol
121
2. It causes considerable tissue shrinkage.
Carnoy’s Fixative
122
3. It is suitable only for small pieces of tissues due to slow penetration.
Carnoy’s Fixative
123
4. It tends to harden tissues excessively and distorts tissue morphology.
Carnoy’s Fixative
124
5. It dissolves fat, lipids, and myelin.
Carnoy’s Fixative : myelin
Ethyl Alcohol
125
6. It leads to polarization unless very cold temperatures (-70°C) are used.
Carnoy’s Fixative
126
7. It dissolves acid-soluble cell granules and pigments.
Carnoy’s Fixative
127
Fixation time: 3 - 4 hours
Clarke’s solution
128
Fixation time: 12 – 24 hours
Alcoholic formalin
129
This is a simple microanatomical fixative made up of saturated
formaldehyde (40%, by weight volume) diluted to 10% with sodium chloride.
10% Formal-Saline
130
This mixture of formaldehyde in isotonic saline was widely used for routine histopathology prior to the introduction of phosphate buffered formalin.
10% Formal-Saline
131
It is recommended for fixation of central nervous tissues and general post-mortem tissues for histochemical examination.
10% Formal-Saline
132
It is also recommended for the preservation of lipids, especially phospholipids.
10% Formal-Saline
133
The buffer tends to prevent the formation of formalin pigment.
10% Formal-Saline
134
Many epitopes require antigen retrieval for successful immunohistochemical (IHC) staining procedure following its use.
10% Formal-Saline
135
Most pathologists feel comfortable interpreting the morphology produced with this type of fixative.
10% Formal-Saline
136
It often produces formalin pigment.
10% Formal-Saline
137
solutions were devised as alternatives to mercuric chloride formulations.
Zinc formalin
138
They are said to give improved results with immunohistochemistry.
Zinc formalin (unbuffered)
139
There are a number of alternative formulas available some of which contain zinc chloride which is thought to be slightly more corrosive than zinc sulphate.
Zinc formalin (unbuffered)
140
has been used on frozen sections and smears.
Clarke’s solution
141
It can produce fair results after conventional processing if fixation time is kept very short.
Clarke’s solution
142
It preserves nucleic acids but extracts lipids.
Clarke’s solution
143
Tissues can be transferred directly into 95% ethanol.
Clarke’s solution
primary fixation specimens: Alcoholic formalin, Formol-acetic alcohol
144
combines a denaturing fixative with the additive and cross-linking effects of formalin.
Alcoholic formalin
145
It is sometimes used during processing to complete fixation following incomplete primary formalin fixation.
Alcoholic formalin
146
It can be used for fixation or post-fixation of large fatty specimens (particularly breast), because it will allow lymph nodes to be more easily detected as it clears and extracts lipids.
Alcoholic formalin
147
If used for primary fixation specimens, it can be placed directly into 95% ethanol for processing.
Alcoholic formalin
Formol-acetic alcohol
148
Fixation time: 1 - 6 hours
Formol-acetic alcohol
149
alcohol is a faster acting agent than alcoholic formalin due to the presence of acetic acid that can also produce formalin pigment.
Formol-acetic alcohol
150
It is sometimes used to fix diagnostic cryostat sections.
Formol-acetic alcohol
151
If used for primary fixation, the specimens can be placed directly into 95% ethanol for processing.
Alcoholic formalin
Formol-acetic alcohol
152
Post-fixation with phenol-formalin for 6 hours or more can enhance immunoperoxidase studies on the tissues, and in some cases, electron microscopy, if it is necessary at a later time to establish a diagnosis.
Gendre's Fixative
153
1. Fixation is faster (fixation time is reduced to one-half).
Gendre's Fixative
154
2. It can be used for rapid diagnosis because it fixes and dehydrates at the same time, e.g., in the frozen section room.
Gendre's Fixative
Methyl Alcohol 100% ; Carnoy’s Fixative
155
3. It is good for preservation of glycogen and for micro-incineration technique (the burning of a minute tissue specimen for identification of mineral elements from the ashes).
Gendre's Fixative
156
4. It is used to fix sputum, since it coagulates mucus.
Gendre's Fixative
157
1. It produces gross hardening of tissues.
Gendre's Fixative
158
2. It causes partial lysis of RBC.
Gendre's Fixative
159
3. Preservation of iron-containing pigments is poor.
Gendre's Fixative
160
4. Formaldehyde does not give as good a morphological picture as glutaraldehyde.
Gendre's Fixative
161
5. Formaldehyde causes little cross-linking under usual fixation conditions where low concentrations of proteins are used, while glutaraldehyde is most effective at cross-linking.
Gendre's Fixative
162
Fixation time: 12-18 hours at 3°C
Newcomer's Fluid
163
1. It is recommended for fixing mucopolysaccharides and nuclear proteins.
Newcomer's Fluid
164
2. It produces better reaction in Feulgen stain than Carnoy's fluid.
Newcomer's Fluid
165
3. It acts both as a nuclear and histochemical fixative.
Newcomer's Fluid
166
PRECIPITATING (ALCOHOLIC) FIXATIVES:
167
METALLIC FIXATIVES:
168
OXIDIZING AGENTS:
169
CHROMATE FIXATIVES:
170
PICRIC ACID FIXATIVES:
171
1. It penetrates and hardens tissues rapidly and well.
Mercuric Chloride
172
2. Nuclear components are shown in fine detail.
Mercuric Chloride
173
3. It precipitates all proteins.
Mercuric Chloride
174
4. It has a greater affinity to acid dyes and is preferred in lieu of formaldehyde for cytoplasmic staining.
Mercuric Chloride
175
5. Trichrome staining is excellent.
Mercuric Chloride
176
6. It is the routine fixative of choice for preservation of cell detail in tissue photography.
Mercuric Chloride
177
7. It permits brilliant metachromatic staining of cells.
Mercuric Chloride
178
8. It is recommended for renal tissue, fibrin, connective tissue and muscle.
Mercuric Chloride
179
1. It causes marked shrinkage of cells (this may be counteracted by addition of acid).
Mercuric Chloride
180
2. It rapidly hardens the outer layer of the tissue with incomplete fixation of the center; therefore, thin sections should be made.
Mercuric Chloride
181
3. Penetration beyond the first 2-3 millimeters is slow; hence, not more than 5 mm. thickness of tissues should be used.
Mercuric Chloride
182
4. If left in fixative for more than 1-2 days, the tissue becomes unduly hard and brittle.
Mercuric Chloride
183
5. It prevents adequate freezing of fatty tissues and makes cutting of frozen tissues difficult.
Mercuric Chloride
184
6. It causes considerable lysis of red blood cells and removes much iron from hemosiderin.
considerable: Mercuric Chloride
Zenker's Solution
185
7. It is inert to fats and lipids.
Mercuric Chloride
186
8. It leads to the formation of black granular deposits in the tissues.
Mercuric Chloride
187
.It reduces the amount of demonstrable glycogen.
Mercuric Chloride
188
10. Compound solutions deteriorate rapidly upon addition of glacial acetic acid to formalin.
Mercuric Chloride
189
11. It is extremely corrosive to metals.
Mercuric Chloride
190
1. Sections must be cleared of mercury deposits before immunostaining.
Black deposits may be removed by adding saturated iodine solution in 95% alcohol, the iodine being decolorized with absolute alcohol in the subsequent stages of dehydration.
Mercuric Chloride
191
2. Compound solutions must always be freshly prepared.
Mercuric Chloride
192
3. The use of metallic forceps and of metal caps to cover the bottles containing the fixative should be avoided.
Mercuric Chloride
193
4. Contact with personal jewelries should be avoided.
Mercuric Chloride
194
5. Mercury-containing solutions (Zenker's or B-5) should always be discarded into proper containers. Mercury, if poured down a drain, will form amalgams with the metal that build up and cannot be removed.
Mercuric Chloride
195
Fixation time: 12-24 hours
Zenker's Solution
10% Formal-Saline
Alcoholic Formalin
196
1. It produces a fairly rapid and even fixation of tissues.
Zenker's Solution
197
2. Stock solutions keep well without disintegration.
Zenker's Solution
198
3. It is recommended for trichrome staining.
Zenker's Solution
199
4. It permits brilliant staining of nuclear and connective tissue fibers.
Zenker's Solution
200
5. It is recommended for congested specimens (such as lung, heart and blood vessels) and gives good results with PTAH and trichrome staining.
Zenker's Solution
201
6. It is compatible with most stains.
Zenker's Solution
202
7. It may act as a mordant to make certain special staining reactions possible.
Zenker's Solution
203
8. It is a stable fixative that can be stored for many years.
Zenker's Solution
204
1. Penetration is poor.
Zenker's Solution
205
2. It is not stable after addition of acetic acid.
Zenker's Solution
206
3. Prolonged fixation (for more than 24 hours) will make tissues brittle and hard.
Zenker's Solution
207
4. It causes lysis of red blood cells and removes iron from hemosiderin.
Zenker's Solution
208
5. It does not permit cutting of frozen sections.
Zenker's Solution
209
6. It has the tendency to form mercuric pigment deposits or precipitates.
Zenker's Solution
210
7. Tissue must be washed in running water for several hours (or overnight) before processing. Insufficient washing may inhibit or interfere with good cellular staining.
Zenker's Solution
211
1. Do not let tissues stay in solution for more than 24 hours.
Zenker's Solution
212
2. Solutions must always be freshly prepared.
Zenker's Solution
213
3. Tissues should be cut thin (2-3 mm.) and hollow organs should be opened to promote complete penetration and fixation.
Zenker's Solution
214
4. Tissues must be washed out thoroughly in running water to permit good staining.
Zenker's Solution
215
5. It produces mercury pigment which should be removed from sections prior to staining and can produce chrome pigment if tissue is not washed
in water prior to processing. After water washing, tissue should be stored in 70% ethanol.
Zenker's Solution
216
6. Mercuric deposits may be removed by immersing tissues in alcoholic iodine solution prior to staining, through a process known as de- zenkerization.
Zenker's Solution
217
Fixation time: 4 – 24 hours
Zenker-Formol (Helly’s) Solution
218
is an excellent fixative for bone marrow, extramedullary hematopoiesis and intercalated discs of cardiac muscle.
Zenker-Formol (Helly’s) Solution
219
However, it produces mercury pigment which should be removed from sections prior to staining and it can produce chrome pigment if tissue is not washed in water prior to processing.
Zenker-Formol (Helly’s) Solution
220
After water washing, fixed tissue should be stored in 70% ethanol.
Zenker-Formol (Helly’s) Solution
221
Because of the low pH of this fixative, formalin pigment may also occur.
Zenker-Formol (Helly’s) Solution
222
Never use metal forceps to handle tissue.
Zenker-Formol (Helly’s) Solution
223
1. It is an excellent microanatomic fixative for pituitary gland, bone marrow and blood containing organs such as spleen and liver.
Zenker-Formol (Helly’s) Solution
224
2. It penetrates and fixes tissues well.
Zenker-Formol (Helly’s) Solution
225
3. Nuclear fixation and staining is better than with Zenker's.
Zenker-Formol (Helly’s) Solution
226
4. It preserves cytoplasmic granules well.
Zenker-Formol (Helly’s) Solution
227
brown pigments are produced if tissues (especially blood containing organs) are allowed to stay in the fixative for more than 24 hours due to RBC lysis. This may be removed by immersing the tissue in saturated alcoholic picric acid or sodium hydroxide.
Zenker-Formol (Helly’s) Solution
228
Fixation time: 4 – 8 hours
Lillie’s B-5 Fixative
229
1. Despite its mercuric chloride content and consequent problems with disposal, this solution is popular for fixation of hematopoietic, bone marrow biopsies and lymphoid tissue.
Lillie’s B-5 Fixative
230
2. Rapid fixation can be achieved in 1 1/2 - 2 hours.
Lillie’s B-5 Fixative
231
3. It produces excellent nuclear detail, provides good results with many special stains, and is recommended for immunohisto-chemical staining.
Lillie’s B-5 Fixative
232
4. Sections will require the removal of mercury pigment prior to staining.
Lillie’s B-5 Fixative
233
5. Tissue should not be stored in this fixative but placed in 70% ethanol instead.
Lillie’s B-5 Fixative
234
6. Over-fixation hardens the tissue and makes cutting difficult.
Lillie’s B-5 Fixative
235
7. The two working solutions are kept separate, since the mixture is unstable. Mix just prior to use.
Lillie’s B-5 Fixative
236
8. Some will form precipitate on standing, but this is of no consequence.
Lillie’s B-5 Fixative
237
9. As with all mercuric chloride fixatives, the mercury pigment can be removed by de-zenkerization.
Lillie’s B-5 Fixative
238
Fixation time: 3-12 hours
Heidenhain's Susa Solution
239
1. It penetrates and fixes tissues rapidly and evenly.
Heidenhain's Susa Solution
240
2. It produces minimum shrinkage and hardening of tissues due to the counter-balance of the swelling effects of acids and the shrinkage effect of mercury.
Heidenhain's Susa Solution
241
3. It permits most staining procedures to be done, including silver impregnation, producing brilliant results with sharp nuclear and cytoplasmic details.
Heidenhain's Susa Solution
242
4. It permits easier sectioning of large blocks of fibrous connective tissues.
Heidenhain's Susa Solution
243
5. may be transferred directly to 95% alcohol or absolute alcohol, thereby reducing processing time.
Heidenhain's Susa Solution
244
is recommended mainly for tumor biopsies especially of the skin; it is an excellent cytologic fixative.
Heidenhain's Susa Solution
245
1. Prolonged fixation of thick materials may produce considerable shrinkage, hardening and bleaching; hence, tissues should not be more than 1 cm. thick.
Heidenhain's Susa Solution
246
2. RBC preservation is poor.
Heidenhain's Susa Solution
247
3. Some cytoplasmic granules are dissolved.
Heidenhain's Susa Solution
248
4. Mercuric chloride deposits tend to form on tissues; these may be removed by immersion of tissues in alcoholic iodine solution.
Heidenhain's Susa Solution
249
5. Weigert's method of staining elastic fibers is not possible
Heidenhain's Susa Solution
250
After using, the tissue should be transferred directly to a high-grade alcohol, e.g. 96% or absolute alcohol, to avoid undue swelling of tissues caused by treatment with low-grade alcohol or water.
Heidenhain's Susa Solution
251
It fixes conjugated fats and lipids permanently by making them insoluble during subsequent treatment with alcohol and xylene.
Fats form hydrated (?), are stained black and therefore are easier to identify.
Osmium Tetroxide (Osmic Acid; OsO4)
252
2. It preserves cytoplasmic structures well, e.g. Golgi bodies and mitochondria.
Osmium Tetroxide (Osmic Acid; OsO4)
253
3. It fixes myelin and peripheral nerves well, hence, it is used extensively for neurological tissues.
Osmium Tetroxide (Osmic Acid; OsO4)
254
4. It produces brilliant nuclear staining with safranin.
Osmium Tetroxide (Osmic Acid; OsO4)
255
5. It adequately fixes materials for ultrathin sectioning in electron microscopy, since it rapidly fixes small pieces of tissues and aids in their staining.
Osmium Tetroxide (Osmic Acid; OsO4)
256
6. It precipitates and gels proteins.
Osmium Tetroxide (Osmic Acid; OsO4)
257
7. It shows uniformly granular nuclei with clear cytoplasmic background.
Osmium Tetroxide (Osmic Acid; OsO4)
258
8. Some tissues (e.g. adrenal glands) are better fixed in vapor form. This eliminates "washing out" of the fixed tissues.
Osmium Tetroxide (Osmic Acid; OsO4)
259
completely permeabilizes cell membranes. The osmolarity of the fixative vehicle or solute is relatively unimportant.
Osmium Tetroxide (Osmic Acid; OsO4)
260
10. It penetrates tissue blocks in a gradient and in large samples the center of the block may not be as well fixed as the peripheral areas.
Osmium Tetroxide (Osmic Acid; OsO4)
261
11. Over-fixation may result in extraction of cell components during dehydration and increases the hardness and brittleness of the tissue (for most tissues, 1mm blocks, should not be exposed to osmium for less than 0.5 or more than 1.5 hours).
Osmium Tetroxide (Osmic Acid; OsO4)
262
1. It is very expensive.
Osmium Tetroxide (Osmic Acid; OsO4)
263
2. It is a poor penetrating agent, suitable only for small pieces of tissues (2-3 mm. thick).
Osmium Tetroxide (Osmic Acid; OsO4)
264
3. It is readily reduced by contact with organic matter and exposure to sunlight, forming a black precipitate which settles at the bottom of the container.
Osmium Tetroxide (Osmic Acid; OsO4)
265
4. Prolonged exposure to acid vapor can irritate the eye, producing conjunctivitis, or cause the deposition of black osmic oxide in the cornea, producing blindness.
Osmium Tetroxide (Osmic Acid; OsO4)
266
5. It inhibits hematoxylin and makes counterstaining difficult.
Osmium Tetroxide (Osmic Acid; OsO4)
267
6. It is extremely volatile.
Osmium Tetroxide (Osmic Acid; OsO4)
268
I. Eyes and skin may be protected by working in a fume hood or wearing protective plastic masks or gloves while using
Osmium Tetroxide (Osmic Acid; OsO4)
269
2. It should be kept in a dark-colored, chemically clean bottle to prevent evaporation and reduction by sunlight or organic matter.
Osmium Tetroxide (Osmic Acid; OsO4)
270
3. It should be kept in a cool place or refrigerated to prevent deterioration.
Osmium Tetroxide (Osmic Acid; OsO4)
271
4. Addition of saturated aqueous mercuric chloride solution (0.5 to 1 ml/100 ml of stock solution) will prevent its reduction with formation of black deposits.
Osmium Tetroxide (Osmic Acid; OsO4)
272
5. Black (?) crystals may be dissolved in cold water.
Osmium Tetroxide (Osmic Acid; OsO4)
273
6. To prevent contact of tissues with black precipitate formed in the bottom of the jar, the tissues may be wrapped in cotton gauze and suspended in the fluid by means of a thread.
Osmium Tetroxide (Osmic Acid; OsO4)
274
7. (?)-fixed tissues must be washed in running water for at least 24 hours to prevent formation of artefacts.
Osmium Tetroxide (Osmic Acid; OsO4)
275
is the most common chrome-osmium acetic acid fixative used, recommended for nuclear preparation of such sections.
Flemming's Solution
276
Fixation time: 24 - 48 bouts
Flemming's Solution
277
1. It is an excellent fixative for nuclear structures, e.g. chromosomes.
Flemming's Solution
278
2. It permanently fixes fat.
Flemming's Solution
279
3. Relatively less amount of solution is required for fixation (less than 10 times the volume of the tissues to be fixed).
Flemming's Solution
280
1. It is a poor penetrating agent; hence, is applicable only to small pieces of tissues.
Flemming's Solution
281
2. The solution deteriorates rapidly and must be prepared immediately before use.
Flemming's Solution
282
3. depress the staining power of hematoxylin (especially Ehrlich's hematoxylin).
Flemming's Solution
283
4. It has a tendency to form artifact pigments; these may be removed by washing the fixed tissue in running tap water for 24 hours before dehydration.
Flemming's Solution
284
5 It is very expensive.
Flemming's Solution
285
- is made up only of chromic and osmic acid, recommended for cytoplasmic structures particularly the mitochondria.
Flemming's solution without acetic acid
286
The removal of acetic acid from the formula serves to improve the cytoplasmic detail of the cell.
Flemming's solution without acetic acid
287
Fixation time: 24 - 48 hours
Flemming's solution without acetic acid
288
Advantages and Disadvantages: same as Flemming's solution.
Flemming's solution without acetic acid
289
is used in 1-2% aqueous solution, usually as a constituent of a compound fixative.
Chromic Acid
290
It precipitates all proteins and adequately preserves carbohydrates.
Chromic Acid
291
It is a strong oxidizing agent; hence, a strong reducing agent (e.g. formaldehyde) must be added to chrome-containing fixatives before use in order to prevent counteracting effects and consequent decomposition of solution upon prolonged standing.
Chromic Acid
292
is used in a 3% aqueous solution.
Potassium Dichromate
293
I. It fixes but does not precipitate cytoplasmic structures.
Potassium Dichromate
294
2. It preserves lipids.
Potassium Dichromate
295
3. It preserves mitochondria (If used in pH 4.5-5.2, mitochondria is fixed. If the solution becomes acidified, cytoplasm, chromatin bodies and chromosomes are fixed but mitochondria are destroyed).
Potassium Dichromate
296
Fixation time: 12-48 hours
Regaud’s (Muller's) Fluid
297
1. It penetrates tissues well.
Regaud’s (Muller's) Fluid
298
2. It hardens tissues better and more rapidly than Orth's fluid.
Regaud’s (Muller's) Fluid
299
3. It is recommended for the demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissues.
Regaud’s (Muller's) Fluid
300
1. It deteriorates and darkens on standing due to acidity; hence, the solution must always be freshly prepared.
Regaud’s (Muller's) Fluid
301
2. Penetration is slow, hence, tissues should not be thicker than 2-3 mm.
Regaud’s (Muller's) Fluid
302
3. Chromate-fixed tissues tend to produce precipitates of sub-oxide, hence should be thoroughly washed in running water prior to dehydration.
Regaud’s (Muller's) Fluid
303
4. Prolonged fixation blackens tissue pigments, such as melanin; this may be removed by washing the tissues in running tap water prior to dehydration.
Regaud’s (Muller's) Fluid
304
5.Glycogen penetration is poor; it is therefore, generally contraindicated for carbohydrates.
Regaud’s (Muller's) Fluid
305
6. Nuclear staining is poor.
Regaud’s (Muller's) Fluid
306
7. It does not preserve fats.
Regaud’s (Muller's) Fluid
307
8. It preserves hemosiderin less than buffered formalin.
Regaud’s (Muller's) Fluid
308
9. Intensity of PAS reaction is reduced.
Regaud’s (Muller's) Fluid
309
Fixation time: 36-72 hours
Orth's Fluid
310
1. It is recommended for study of early degenerative processes and tissue necrosis.
Orth's Fluid
311
2. It demonstrates rickettsiae and other bacteria.
Orth's Fluid
312
3. It preserves myelin better than buffered formalin.
Orth's Fluid
313
Disadvantages: Same as in Regaud's fluid.
Orth's Fluid
314
The complementary effects of the three ingredients work well together to maintain morphology.
Bouin's Solution
315
Specimens are usually fixed for 24 hours.
Bouin's Solution
316
Prolonged storage in this acidic mixture causes hydrolysis and loss of stainable DNA and RNA.
Bouin's Solution
317
Thorough washing after fixation is necessary.
Bouin's Solution
318
is recommended for fixation of embryos and pituitary biopsies.
Bouin's Solution
319
It gives very good results with tissue that is subsequently stained with trichrome.
Bouin's Solution
320
It preserves glycogen well but usually lyses erythrocytes.
Bouin's Solution
321
It is sometimes recommended for gastro-intestinal tract biopsies, animal embryos and endocrine gland tissue.
Bouin's Solution
322
It stains tissue bright yellow due to picric acid.
Bouin's Solution
323
Excess picric should be washed from tissues prior to staining with 70% ethanol.
Bouin's Solution
324
Because of its acidic nature, it will slowly remove small calcium deposits and iron deposits.
Bouin's Solution
325
1. It is an excellent fixative for glycogen demonstration.
Bouin's Solution
326
2. It penetrates tissues well and fixes small tissues rapidly.
Bouin's Solution
327
3. The yellow stain taken in by tissues prevents small fragments from being overlooked.
Bouin's Solution
328
4. It allows brilliant staining with the trichrome method.
Bouin's Solution
329
5. It is suitable for Aniline stains (Mallory's, Heidenhain's or Masson's methods).
Bouin's Solution
330
6. It precipitates all proteins.
Bouin's Solution
331
7. It is stable.
Bouin's Solution
332
1. It causes RBC hemolysis and reduces the amount of demonstrable ferric iron in tissue.
Bouin's Solution
333
2. It is not suitable for frozen sections because it causes frozen sections to crumble when cut.
Bouin's Solution
334
3. Prolonged fixation makes tissues hard, brittle and difficult to section. Tissues should not be allowed to remain in the fluid for more than 12-24 hours (depending on size).
Bouin's Solution
335
4. Picrates form protein precipitates that are soluble in water; hence, tissues must be first rendered insoluble by direct immersion in 70% ethyl alcohol.
Bouin's Solution
336
5. Picric acid fixed tissues must never be washed in water before dehydration.
Bouin's Solution
337
6. Picric acid will produce excessive staining of tissues; to remove the yellow color, tissues may be placed in 70% ethyl alcohol followed by 5% sodium thiosulfate and then washed in running water.
Bouin's Solution
338
7. Picric acid is highly explosive when dry, and therefore must be kept moist with distilled water or saturated alcohol at 0.5 to 1% concentration during storage.
Bouin's Solution
339
8. It alters and dissolves lipids.
Bouin's Solution
340
9. It interferes with Azure eosin method of staining; hence, tissues should be thoroughly washed with alcohol.
Bouin's Solution
341
It is recommended for gastro-intestinal tract specimens and fixation of endocrine tissues.
Hollande’s Solution
342
It produces less lysis than Bouin’s Solution.
Hollande’s Solution
343
It has some decalcifying properties.
Hollande’s Solution
344
The fixative must be washed from tissues if they are to be put into phosphate buffered formalin on the processing machine because an insoluble phosphate precipitate will form.
Hollande’s Solution
345
Fixation time: 4 – 18 hours
Bouin's Solution
Hollande’s Solution
Gendre’s solution
346
Store at room temperature
Bouin's Solution
347
This is an alcoholic Bouin’s solution that appears to improve upon ageing.
Gendre’s solution
348
It is highly recommended for the preservation of glycogen and other carbohydrates.
Gendre’s solution
349
After fixation the tissue is placed into 70% ethanol.
Gendre’s solution
350
Residual yellow color should be washed out before staining.
Gendre’s solution
351
1. It produces minimal distortion of micro-anatomical structures and can be used for general and special stains. (The shrinking effect of picric acid is balanced by the swelling effect of glacial acetic acid.)
Gendre’s solution
352
2. It is an excellent fixative for preserving soft and delicate structures (e.g. endometrial curettings).
Gendre’s solution
353
3. It penetrates rapidly and evenly, and causes little shrinkage.
Gendre’s solution
354
4. Yellow stain is useful when handling fragmentary biopsies.
Gendre’s solution
355
5. It permits brilliant staining of tissues.
Gendre’s solution
356
6. It is the preferred fixative for tissues to be stained by Masson's trichrome for collagen, elastic or connective tissue. (If tissue is fixed in formalin, a pre-treatment in Bouin’s solution (as mordant prior to trichrome stain) is recommended.
Gendre’s solution
357
7. It preserves glycogen.
Gendre’s solution
358
8. It does not need "washing out".
Gendre’s solution
359
1. It penetrates large tissues poorly; hence, its use is limited to small fragments of tissue.
Gendre’s solution
360
2. Picrates are soluble in water; hence, tissues should not be washed in running water but rather transferred directly from fixative to 70% alcohol.
Gendre’s solution
361
3. It is not suitable for fixing kidney structures, lipid and mucus.
Gendre’s solution
362
4. It destroys cytoplasmic structures, e.g. mitochondria.
Gendre’s solution
363
5. It produces RBC hemolysis and removes demonstrable ferric iron from blood pigments.
Gendre’s solution
364
6. It reduces or abolishes Feulgen reaction due to hydrolysis of nucleoproteins.
Gendre’s solution
365
1. It fixes and precipitates nucleoproteins.
GLACIAL ACETIC ACID
366
2. It precipitates chromosomes and chromatin materials; hence, is very useful in the study of nuclear components of the cell. In fact, it is an essential constituent of most compound nuclear fixatives.
GLACIAL ACETIC ACID
367
3. It causes tissues (especially those containing collagen) to swell. This property is used in certain compound fixatives to counteract the shrinkage produced by other components (e.g. mercury).
GLACIAL ACETIC ACID
368
1. When combined with Potassium Dichromate, the lipid-fixing property of the latter is destroyed (e.g. Zenker's fluid).
GLACIAL ACETIC ACID
369
2. It is contraindicated for cytoplasmic fixation since it destroys mitochondria and Golgi elements of cells.
GLACIAL ACETIC ACID
370
3. Concentrated acetic acid is corrosive to skin and must, therefore, be handled with appropriate care, since it can cause skin burns, permanent eye damage, and irritation to the mucous membranes. These burns or blisters may not appear until hours after exposure.
GLACIAL ACETIC ACID
371
4. Latex gloves offer no protection, so especially resistant gloves, such as those made of nitrile rubber, are worn when handling the compound.
GLACIAL ACETIC ACID
372
1. It is recommended for acid mucopolysaccharides.
LEAD FIXATIVES
373
2. It fixes connective tissue mucin.
LEAD FIXATIVES
374
It takes up C02 to form insoluble lead carbonate especially on prolonged standing. This may be removed by filtration or by adding acetic acid drop by drop to lower the pH and dissolve the residue.
LEAD FIXATIVES
375
1. It precipitates proteins.
TRICHLOROACETIC ACID
376
2. Its marked swelling effect on tissues serves to counteract shrinkage produced by other fixatives.
TRICHLOROACETIC ACID
377
3. It may be used as a weak decalcifying agent.
TRICHLOROACETIC ACID
378
4. Its softening effect on dense fibrous tissues facilitates preparation of such sections.
TRICHLOROACETIC ACID
379
It is a poor penetrating agent, hence, is suitable only for small pieces of tissues or bones.
TRICHLOROACETIC ACID
380
1. It is recommended for the study of water diffusible enzymes especially phosphatases and lipases.
ACETONE
381
2. It is used in fixing brain tissues for diagnosis of rabies.
ACETONE
382
3. It is used as a solvent for certain metallic salts to be used in freeze substitution techniques for tissue blocks.
ACETONE
383
1. It produces inevitable shrinkage and distortion.
ACETONE
384
2. It dissolves fat.
ACETONE
385
3. It preserves glycogen poorly.
ACETONE
386
4. It evaporates rapidly.
ACETONE
387
provides a stable medium for transport of fresh unfixed tissues, such as renal, skin and oral mucosa biopsies, which will undergo subsequent frozen section and immunofluorescence studies.
MICHEL’S SOLUTION
388
Medium is not suitable for transporting cells for flow cytometry or for tissues used for fluorescent in-situ hybridization (FISH).
MICHEL’S SOLUTION
389
It is not a fixative, and is not suitable for any other use (particularly, for transporting living cells for flow cytometry).
MICHEL’S SOLUTION
390
It should be kept refrigerated (not frozen) until use.
MICHEL’S SOLUTION
391
Specimens may be kept in it at room temperature for 5 days while in transport until they can be delivered to the reference laboratory.
MICHEL’S SOLUTION
392
This simple salt solution maintains pH, but does not kill most pathogens.
MICHEL’S SOLUTION
393
Specimens received in transport medium should be washed in three changes of washing solution (10 minutes for each wash).
MICHEL’S SOLUTION
394
It is not suitable for FISH studies.
MICHEL’S SOLUTION
395
Adjust pH to 7.4
10% Neutral-Buffered Formalin
396
1. It prevents precipitation of acid formalin pigments on postmortem tissue.
10% Neutral-Buffered Formalin
397
2. It is the best fixative for tissues containing iron pigments and for elastic fibers which do not stain well after Susa, Zenker’s or
chromate fixation.
10% Neutral-Buffered Formalin
398
3. It requires no post-treatment after fixation and goes directly into 80% alcohol for processing.
10% Neutral-Buffered Formalin
399
1. It is longer to prepare; hence, is time-consuming.
10% Neutral-Buffered Formalin
400
2. Positivity of mucin to PAS is reduced.
10% Neutral-Buffered Formalin
401
3. It may produce gradual loss in basophilic staining of cells.
10% Neutral-Buffered Formalin
402
4. Reactivity of myelin to Weigert's iron hematoxylin stain is reduced.
10% Neutral-Buffered Formalin
403
5. It is inert towards lipids, especially neutral fats and phospholipids.
10% Neutral-Buffered Formalin
404
They are said to give improved results with immunohistochemistry.
Zinc formalin (unbuffered)
405
There are a number of alternative formulas available some of which contain zinc chloride which is thought to be slightly more corrosive than zinc sulphate.
Zinc formalin (unbuffered)
406
There is no need for "washing-out".
Formalin
Formol-Corrosive (Formol-Sublimate)
407
Tissues can be transferred directly from fixative to alcohol.
Formol-Corrosive (Formol-Sublimate)
