Lec 2: Tissue Processing 1 & 2 Flashcards
(57 cards)
1
Q
The second step in manual tissue processing
A
Dehydration
2
Q
The process of removing intracellular and extracellular water from the tissue
A
Dehydration
3
Q
Characteristics of an ideal dehydrating solution (7)
A
- Should dehydrate properly/rapidly without causing shrinkage or distortion
- Should dehydrate even fatty tissues
- Should NOT evaporate quickly
- Should NOT harden tissue excessively
- Should NOT remove stains
- Should NOT be toxic to the body
- Should NOT be a fire hazard
4
Q
Dehydrating agent should NOT be less than ___ times the volume of the tissue.
A
10
5
Q
Commonly used dehydrating agents (6)
A
- alcohol
- acetone
- cellosolve
- dioxane
- triethyl phosphate
- tetrahydrofuran
6
Q
- clear, colorless, flammable
- for routine dehydration
- not poisonous
- fast-acting
- penetrates tissue easily
- mixed with water and other organic solvents.
A
Ethyl Alcohol
• best dehydrating agent
7
Q
- toxic dehydrating agent
- for blood & tissue film
- for smear preparations
A
Methyl Alcohol
8
Q
- slow dehydrating agent
› doesn’t require rapid processing - utilized in plant and animal micro-techniques
- less shrinkage & hardening
A
Butyl Alcohol
9
Q
ACETONE
1. boiling point
2. Dehydrates how long?
3. Can tissues be placed in it for a prolonged period of time?
4. How well does it penetrate the tissue?
A
- 56°C
- 30 minutes to 2 hours
- NO, because it causes brittleness in tissues.
- Penetrates poorly
10
Q
- clear and colorless
- rapid-acting
- for most urgent biopsies
- extremely volatile & flammable
- mixes with water, ethanol, and most organic solvents
A
Acetone
11
Q
- rapid-acting
- combustible (110-120°F) & toxic
- tissue can be stored for months without hardening or distorting.
A
Cellosolve
12
Q
CELLOSOLVE
1. boiling point
2. Other name
A
- 156.4°C
- Ethylene glycol monoethyl ether
13
Q
The dehydrating agent wherein the reproductive, fetal, urinary, and blood systems are particularly vulnerable to its toxic side effects
A
Cellosolve
14
Q
DIOXANE
1. boiling point
2. Refractive index
3. Other name
A
- 101.5°C
- 1.42
- Diethylene dioxide
15
Q
- readily miscible with water, melted paraffin, alcohol, and xylol
- tissues can be left for a long time without affecting the staining properties
A
Dioxane
16
Q
- miscible in water & paraffin
- can dissolve many substances including fats
- for demixing, clearing, and dehydrating paraffin sections
- odorous
- evaporates rapidly
A
Tetrahydrofuran
17
Q
TRIETHYL PHOSPHATE
1. Boiling point
2. Soluble in what substances
A
- 215°C
- Alcohol, water, ether, benzene, Chloroform, acetone, xylene
18
Q
- used to dehydrate sections and smears
- minimum shrinkage
- removed water readily
- little distortion and hardening
A
Triethylphosphate
19
Q
The killing, penetration, and hardening of tissues
A
Fixation
- killing: stops metabolic processes
- penetration: fixative penetrates tissue
- hardening: for easier trimming & cutting
20
Q
First and most critical step involving preserving fresh tissue
A
Fixation
21
Q
The alteration of tissues by stabilizing proteins so that the tissues become resistant to further changes
A
Fixation
22
Q
Primary Aim of Fixation
A
To preserve the morphologic and chemical integrity of the cell
23
Q
Secondary Aim of Fixation
A
To harden and protect the tissue from trauma
24
Q
The functions of fixation
A
- to preserve the tissue
- to prevent breakdown of cellular elements
- to coagulate/precipitate protoplasmic substances
25
The practical considerations to optimize fixation of tissue (8)
- duration of fixation
- osmolality
- concentration
- penetration
- volume
- speed
- temperature
- thickness
26
**Practical Considerations:**
1. Specimen should be fixed ___ hour/s from removal.
2. Fixative must be ___ times the size of the tissue
3. Ratio of fixative to tissue.
4. Formalin diffuses into tissue approximately __mm/hr.
5. Duration of fixation for LM & EM.
1. Specimen should be fixed _at least 1_ hour/s from removal.
2. Fixative must be _20_ times the size of the tissue
3. **20:1 or 10:1**
4. Formalin diffuses into tissue approximately _1mm/hr_.
5. Duration of fixation
a. LM: _2/4–6 hours_
b. EM: _3 hours_
27
**Practical Considerations:**
6. Osmolality
a. LM:
b. EM:
c. 10% NBF:
7. Concentration
a. Formaldehyde:
b. Glutaraldehyde:
8. Temperature
a. Routine manual:
b. Routine automated:
c. EM:
d. DNA:
e. RNA:
f. microwave:
9. Thickness of tissue
a. LM:
b. EM:
6. Osmolality
a. LM: _slightly hypertonic (400-450 mOsm)_
b. EM: _isotonic (340 mOsm)_
c. 10% NBF: _1500 mOsm_
7. Concentration
a. Formaldehyde: _10%_
b. Glutaraldehyde: _3%; 0.25%_
8. Temperature
a. Routine manual: _20-22°C_
b. Routine automated: _40°C_
c. EM: _0-40°C_
d. DNA: _65°C_
e. RNA: _45°C_
f. microwave: _65°C_
9. Thickness of tissue
a. LM: _2cm_
b. EM: _1-2mm_
28
Characteristics of a Good Fixative (8)
- cheap & economical
- stable and safe to handle
- fast-acting & even penetration
- inhibits bacterial decomposition & autolysis
- at least *isotonic*
- hardens tissue
- renders tissue *insensitive to subsequent processing*
- is compatible with many staining procedures
29
1. What is a simple fixative?
2. Give examples.
1. Made up of only 1 component substance
2. Examples:
a. Aldehyde
b. Metallic fixative
c. Picric Acid
d. Acetic acid
e. Acetone
f. Alcohol
g. Osmium tetroxide
30
1. What is a Compound fixative?
2. Give examples
1. Made up of 2 or more fixatives added together
2. Examples:
a. Zenker's Fluid
b. Bouin's fluid
c. Formol saline
31
Fixatives under Aldehyde Fixative
- formaldehyde (formalin)
- 10% formol saline
- 10% NBF
- Formol-corrosive
- alcoholic formalin
- glutaraldehyde
- formol calcium
- Karnovsky's Paraformaldehyde Glutaraldehyde
- Acrolein
32
- buffered at pH 7 to 8
- fixed tissue by *forming a cross linkage*
Formaldehyde (formalin)
33
- for fixation of *CNS tissues* and *_general_ post mortem tissue*
- made up of 40% formaldehyde + NaCl + distilled water
- fixation time: 12 to 24 hours
10% formol saline
34
**10% Formol Saline**
1. Fixation time
2. Composition
1. 12 to 24 hours
2. 40% formaldehyde + NaCl+ distilled water
35
- for preservation & storage of surgical, post mortem, and research specimens
- best for tissues containing *iron pigments & elastic fibers*
10% NBF
36
**10% NBF**
Fixation time
4 to 24 hours
37
- corrosive
- for *routine* post-mortem tissues
- fixation time: 3 to 24 hours
Formol-corrosive
38
**Formol-corrosive**
Fixation time
3 to 24 hours
39
- denaturing fixative with the additive in the cross links effect of formalin
- for rapid diagnosis
- for preservation of glycogen & *micro-incineration*
- used to fix sputum and coagulate *mucus*
Alcoholic Formalin
40
**Alcoholic Formalin**
1. Post fixation for ___ hours enhances the immunoperoxidase studies on tissues.
2. Other name.
1. **6 hours or more**
2. Gendre's Fixative
41
**Glutaraldehyde**
1. Composition
2. Fixation time
1. 2 formaldehyde linked by 3 carbon chains
2. 1½ hour to 2 hours
42
- for adipose tissue (phospholipid)
Formol Calcium
43
- surgical biopsy
Acrolein
44
Examples of the fixatives in _Mercuric chloride_ under Metallic Fixative
- Zenker's Fluid
- Zenker-formol (Helly's solution)
- Hedenhain's Susa
- B-5 Fixative
45
- for tissue photography: renal tissues, fibrin, CT and muscles
- ADVANTAGE:
» best for hematopoietic & reticuloendothelial tissues
- DISADVANTAGE:
» rapidly hardens outer layer of tissue
Mercuric Chloride
• most common metallic fixative
46
- used to prevent *turbidity* and formation of *dark precipitate*
Zenker's Fluid
47
**Zenker's Fluid**
1. Composition
2. Fixation time
1. Mercuric chloride + Glacial acetic acid
2. 12 to 24 hours
48
- excellent for *bone marrow, extra medullary hematopoiesis, and intercalated disc of the cardiac muscle.*
Zenker-formol (Helly's)
49
**Zenker-formol**
1. Fixation time
2. Other name
1. 4 to 24 hours
2. Helly's solution
50
**Hedenhain's Susa**
1. Recommended for ___?
2. Fixation time
1. For tumor biopsy especially the skin
2. 3 to 12 hours
51
- for bone marrow biopsy
- enhances nuclear detail to identify normal & abnormal cell types
B-5 Fixative
52
**B-5 Fixative**
1. Fixation time
1. 4 to 8 hours
53
Chromate acid preserves ___
Carbohydrates
54
- For the demonstration of:
» chromatin
» mitochondria
» Golgi bodies
» RBC
Regaud's Fluid
55
**Regaud's Fluid**
1. Tissue should NOT be thicker than ____
2. Fixation time
3. Other name
1. **2-3 mm**
2. 12 to 48 hours
3. Muller's solution
56
**Potassium Dichromate**
1. Preserves ___
2. pH range
1. Preserves lipids and mitochondria
2. to preserve mitochondria, pH should be _4.5 to 5.2_
57
- recommended for the study of _early degenerative processes_ and _tissue necrosis_
- for demonstration of rickettsia bacteria and preserves better myelin
- Fixation time: 36 to 72 hours
Orth's fluid
