FIXATION Flashcards
1
Q
The first and most critical step in histotechnology
A
Fixation
2
Q
PRIMARY AIM OF FIXATION:
A
to preserve the morphologic and chemical integrity of the cell in as lifelike manner as possible
3
Q
SECONDARY GOAL OF FIXATION:
A
to harden and protect the tissue from the trauma of further handling, so that it is easier to cut during gross examination
4
Q
Satisfactory fixation occurs between the pH of
A
pH 6 and 8
5
Q
Fixation of surgical specimens is traditionally carried out at
A
room temperature
6
Q
Many laboratories use tissue processors that work at
A
40°C
7
Q
Formalin heated to ______ is sometimes used for the rapid fixation of very urgent biopsy specimens, although the risk of tissue distortion is increased
A
60°C
8
Q
Can be used to fix tissues with tuberculosis
A
Formalin at 100°C
9
Q
Brain is usually suspended whole in ____________ for 2-3 weeks to ensure fixation and some hardening prior to sectioning
A
10% buffered formalin
10
Q
For fixation, the best results are usually obtained using what type of solution?
A
slightly hypertonic solutions (400-450 mOsm)
11
Q
Causes polymerization of the aldehyde, with consequent decrease in its effective concentration
A
presence of a buffer
12
Q
This have been found to be an ideal concentration for immuno-electron microscopy
A
Low concentrations of glutaraldehyde (0.25%)
13
Q
Primary fixation using buffered formalin should be for how hours?
A
2-6 hours
14
Q
The maximum effectiveness of fixation is noted to be _____ the tissue volume
A
20 times
15
Q
Traditionally, the amount of fixative used has been ________the volume of tissue to be fixed.
A
10-25 times
16
Q
What is the adequate fixation time?
A
4 - 6 hours
17
Q
What is the recommended size of the tissue?
A
2 cm2 and no more than 4 mm thick
18
Q
Used to slow down decomposition if the tissue needs to be photographed and cannot be fixed immediately
A
Refrigeration
19
Q
Continues to undergo mitosis (growth) up to 30 minutes after death when refrigerated
A
bone marrow
20
Q
This deteriorate very quickly
A
Brain cells
21
Q
Are made up of only one
component substance
A
Simple Fixatives
22
Q
Are those that are made up of two or more fixatives which have been added together to obtain the optimal combined effect of their individual actions upon the cells and tissue constituents.
A
Compound Fixatives
23
Q
Are those that permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question
A
Microanatomical Fixatives
24
Q
Enumerate the fixatives under the aldehydes of Simple fixatives
A
- Formaldehyde
- Glutaraldehyde
25
Enumerate the fixatives under metallic fixatives of simple fixatives
- Mercuric chloride
- Chromate fixatives ( potassium dichromate, and chromic acid)
- Lead fixatives (Picric acid, acetic acid, acetone alcohol, and osmium teroxide or osmic acid)
- Heat
26
Enumerate the fixatives under microanatomical fixatives
- 10% Formol saline
- 10% Neutral buffered formalin
- Heidenhain’s Susa
- Formol sublimate (formol corrosive)
- Zenker’s solution
- Zenker-formol (Helly's solution)
- Bouin's solution
-Brasil's solution
27
Enumerate the fixatives that have a ph of 4.6 or less ( hint: Nuclear fixatives)
- Flemming's fluid
- Carnoy's fluid
- Bouin's fluid
- Newcomer's fluid
- Heidenhain's fluid
28
Enumerate the fixatives that have a pH of more than 4.6 (hint: Cytoplasmic fixatives)
- Flemming's fluid without acetic acid
- Kelly's fluid
- Formalin with “post-chroming”
- Regaud's fluid (Muller's fluid)
- Orth's fluid
29
These fixatives preserve the chemical constituents of cells and tissues
Histochemical fixatives
30
Enumerate the fixatives under histochemical fixatives
- Formol saline 10% or 10% Formol Saline
- Absolute Ethyl alcohol
- Acetone alcohol
- Newcomer's fluid
31
For lipid fixation, fixatives that contain ___________ and _________ can be effective for preservation of lipids in cyrostat sections
mercuric chloride and potassium dichromate, respectively
32
These fixatives are generally recommended for **glycogen fixation**
Alcoholic fixatives
33
These are the most commonly used fixatives for amino acid histochemistry
Neutral buffered formol saline or formaldehyde vapor
34
The most useful fixatives for preserving glycogen are alcohol-based such as
Rossman's fluid or cold absolute alcohol
35
One of the most widely used fixatives is________ **which is made from formaldehyde**, a gas produced by the oxidation of methyl alcohol, and is soluble in water to the extent of 37-40% weight in volume
10% formalin
36
A simple microanatomical fixative made up of saturated formaldehyde (40% by weight volume) diluted to 10% with sodium chloride.
10% Formol- Saline
37
It is recommended for fixation of **central nervous tissues and general post-mortem tissues** for histochemical examination.
10% Formol-Saline
38
Recommended for **preservation and storage** of surgical, post-mortem and research specimens.
10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH 7)
39
This aldehyde fixative is recommended for **routine** post-mortem tissues.
Formol-Corrosive (Formol-Sublimate)
40
Post-fixation with ________ 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.
phenol-formalin (Alcoholic Formalin/ Gendre's Fixative)
41
It acts in a manner **similar to formaldehyde** and is sometimes utilized for routine light microscopic work.
Glutaraldehyde
42
Enumerate the six fixatives under Aldehyde Fixative
- Formaldehyde (Formalin)
- 10% Formol-Saline
- 10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH 7)
- Formol-Corrosive (Formol-Sublimate)
- Alcoholic Formalin (Gendre’s) Fixative
- Glutaraldehyde
43
Enumerate the fixatives under Mercuric Chloride (Metallic fixative)
- Zenker's fluid
- Zenker-formol (Helly's solution)
- Heidenhain's Susa solution
- B-5 Fixative
44
Buffered glutaraldehyde, followed by secondary fixation in _________ is satisfactory for electron microscopy.
osmium tetroxide
45
The most common metallic fixative, frequently used in saturated aqueous solutions of 5-7%; it is included in many compound fixatives
Mercuric Chloride
46
Widely used as a secondary fixative reacting with a number of amino acid residues and accompanied by **spectroscopic changes**, probably due to reaction with **histidine residues.**
Mercuric Chloride
47
Made up of **mercuric chloride stock solution** to which glacial acetic acid has been added just before its use to prevent turbidity and formation of a dark precipitate.
Zenker's fluid
48
It is recommended for fixing **small pieces of liver, spleen, connective tissue fibers** and **nuclei**.
Zenker's fluid
49
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)
50
Recommended mainly for **tumor biopsies** especially of the **skin**; it is an excellent cytologic fixative.
Heidenhain's Susa Solution
51
Commonly used for **bone marrow biopsies**.
B-5 fixative
52
Used in 1-2% aqueous solution, usually as a constituent of a compound fixative. 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
53
Preserves lipids and mitochondria
Potassium Dichromate
54
It is recommended for demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissues.
Regaud’s (Muller’s) Fluid
55
Recommended for study of **early degenerative processes** and **tissue necrosis.** It demonstrates **rickettsiae** and other bacteria.
Orth's fluid
56
Recommended for acid mucopolysaccharides. It fixes connective tissue mucin.
Lead fixatives
57
Enumerate the fixatives under Chromate fixatives
- Chromic acid
- Potassium Dichromate
- Ragaud's /Ragard's (huhuhu) (Muller's fluid)
- Orth's fluid
58
Enumerate the fixatives under Picric acid fixatives
- Bouin's solution
- Brasil's Alcoholic Picroformol Fixative
59
Normally **used in conjunction with other fixatives** to form a compound solution.
Acetic acid
60
It **fixes and precipitates nucleoproteins**. It precipitates chromosomes and chromatin materials.
Glacial Acetic acid
61
**Rapidly denatures** and precipitates proteins by destroying hydrogen and other bonds
Alcohol (Alc. fixatives)
62
Can be used to fix and preserve **glycogen**, **pigments**, blood, tissue films and smears.
Absolute alcohol
63
It is excellent for fixing dry and wet smears, **blood smears and bone marrow tissues**. It **fixes and dehydrates** at the same time.
Methyl Alcohol 100%
64
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%
65
**Used at concentrations of 70-100%**. If lower concentrations are used, the RBC’s become hemolyzed and WBC’s are inadequately preserved. It may be used as a simple fixative.
Ethyl alcohol
66
Recommended for fixing chromosomes, lymph glands and urgent biopsies.
Carnoy’s Fluid
67
Rapid in action and may be used for urgent biopsy specimens for paraffin processing within 5 hours
Carnoy’s Fluid
68
Enumerate the fixatives under Alcohol fixative
- Methyl Alcohol 100%
- Isopropyl Alcohol 95%
- Ethyl alcohol
- Carnoy's fluid
- Newcomer's fluid
69
This is a **pale yellow powder** which dissolves in water (up to 6% at 20°C) to form a strong oxidizing solution.
Osmium tetroxide (Osmic acid)
70
Most common **chrome-osmium acetic acid** fixative used, recommended for nuclear preparation of such sections
Flemming's solution
71
Made up only of chromic and osmic acid, recommended for cytoplasmic structures particularly the mitochondria
Flemming’s solution without acetic acid
72
IDENTIFY:
- It precipitates proteins.
- Its marked swelling effect on tissues serves to counteract shrinkage produced by other fixatives.
- It may be used as a weak decalcifying agent.
- Its softening effect on dense fibrous tissues facilitates preparation of such sections.
Trichloroacetic acid
73
Used at **ice cold temperature** ranging from -5°C to 4°C
Acetone
74
Enumerate the fixatives under Osmium Tetroxide (Osmic acid)
- Flemming's solution
- Flemming's solution without acetic acid
75
This procedure involves thermal coagulation of tissue proteins for rapid diagnosis, usually employed for frozen tissue sections and preparation of bacteriologic smears.
Heat fixation
76
This may be done before DEHYDRATION and on DEPARAFFINIZED sections before staining
Secondary fixation
77
A **form of secondary fixation** whereby a primarily fixed tissue is placed in aqueous solution of 2.5 - 3% potassium dichromate for 24 hours to act as mordant for better staining effects and to aid in cytologic preservation of tissues.
Post- Chromatization
78
The process of removing excess fixative from the tissue after fixation in order to improve staining and remove artefacts from the tissues.
Washing-out
79
Used to remove:
a. excess chromates from tissues fixed in Kelly's, Zenker’s, and Flemming’s solutions
b. excess formalin
c. excess osmic acid
Tap water
80
Used to wash out excess amount of **picric acid** (Bouin's solution).
50-70% alcohol
81
Used to remove excessive **mercuric fixatives.**
Alcoholic iodine
82
Prevents complete penetration of fixative; hence, tissues that contain this are fixed slowly and poorly
Presence of mucus
83
This inactivates enzymes
Cold temperature
84
Tissues containing large amount of blood (e.g. blood vessels and spleen) should be flushed out with?
Saline
85
This require less fixative and shorter fixation time.
Smaller and thinner tissues
85
Accelerates fixation but hastens autolytic changes and enzyme destruction
Moderate heat (37-56°C)
86
A well-known artefact that may be produced under **acid conditions**
Formalin pigment
87
May be found in surgical specimens particularly in liver biopsies, associated with an intense eosinophilic staining at the center of the tissue in H &E stained sections.
Crush artefact
88
Works as a physical agent similar in mechanism to vacuum, oven (heat) and agitation to increase the movement of molecules and accelerate fixation
Microwave technique
89
Enumerate the factors that affect fixation of tissues
Retarded by:
- Size and thickness of the tissue specimen
- Presence of mucus
- Presence of fat
- Presence of blood
Enhanced by:
- Size and thickness of tissue
- Agitation
90
Thorough rinsing out of formalin for tissue less than 1 mm thick: Isopropanol
1 change, 3 minutes
90
Thorough rinsing out of formalin for tissue less than 1 mm thick: Ethanol
1 change, 5 minutes
91
Thorough rinsing out of formalin for tissue less than 1 mm thick: Paraffin
2 changes; 1 at 67°C for 2 minutes; second change at 82°C for 5 minutes
91
Thorough rinsing out of formalin for tissue 1-2 mm thick: Ethanol
15 minutes
92
Thorough rinsing out of formalin for tissue 1-2 mm thick: Isopropanol
15 minutes
93
Thorough rinsing out of formalin for tissue 1-2 mm thick: Paraffin
- 1 change — 67°C for 10 minutes
- 1 change — 82°C for 12 minutes
94
Thorough rinsing out of formalin for tissue 2-5 mm thick: Paraffin
30 minutes at 67°C; 60 minutes at 82°C
95
Thorough rinsing out of formalin for tissue 2-5 mm thick: Ethanol
60 minutes
95
Thorough rinsing out of formalin for tissue 2-5 mm thick: Isopropanol
45 minutes
96
Its gneral aim is to preserve the maximum enzyme activity at its original localization, while also preserving structural integrity.
Enzyme histochemistry
97
What are the most useful primary fixatives for electron microscopy?
- osmium tetroxide
- glutaraldehyde
- paraformaldehyde,
with the whole procedure performed at 4°C.
98
Microwave energy interacts with dipolar molecules, causing oscillation at a frequency of
2450 mHz
98
For routine studies in electron microscopy the fixatives used are
Glutaraldehyde or osmium
tetroxide
99
For electron histochemistry and electron immunocytochemistry in electron microscopy, the fixative used is?
Karmnovsky's paraformaldehyde-glutaraldehyde
100
What are the usual primary fixative used in secondary fixation
10% formalin or 10% formol saline
101
When tissue is fixed in 10% buffered neutral formalin, it may require secondary fixation with ___________ which acts as a mordant
Zenker's solution
102
IDENTIFY the specific use/target of the following stains:
a. Masson's trichome
b. Mallory's aniline blue
c. Phosphotungstic acidhematoxylin (PTAH) stain
a. connective tissue
b. collagen
c. striated muscle
103
Fixative used for post- fixation in electron microscopy
Osmium tetroxide
