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Histopathology Laboratory > Fixation > Flashcards

Fixation Flashcards

1
Q

What are the two mechanisms of fixation?

A

Additive: Crosslinks proteins (e.g., formalin)

Non-additive: Dehydration (e.g., alcohol)

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2
Q

What are the ideal conditions for routine light microscopy?

A

10% neutral buffered formalin

pH 6-8

20:1 fixative:tissue ratio

Room temperature

4mm max thickness

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3
Q

What fixation is used for:

EM studies?

TB specimens?

Nucleic acid preservation?

A

3% glutaraldehyde (0-4°C)

100°C for 30 min

RNA: 45°C; DNA: 65°C

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4
Q

What causes:

Cell shrinkage

Antigen masking

A

Hypertonic solutions (>450 mOsm)

Prolonged formalin fixation (>24hr)

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5
Q

What are key timing considerations?

A

Primary fixation: 6-24 hours

Formalin penetrates 1mm/hour

EM fixation: 3 hours max

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6
Q

Match fixatives to their use:

Formalin

Osmium tetroxide

Ethanol

A

Routine histology

Lipid preservation (EM)

Cytology smears

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7
Q

What are 4 key benefits of proper tissue fixation?

A

Prevents cellular distortion

Inhibits bacterial decomposition

Enhances staining contrast

Reduces infection risk
(Bonus: Acts as mordant for certain stains)

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8
Q

Compare:

Simple vs compound fixatives

Microanatomical vs cytological fixatives

A

Simple: 1 component (e.g., formalin); Compound: Multiple components

Microanatomical: Preserves tissue architecture; Cytological: Targets specific cell components

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9
Q

How do nuclear and cytoplasmic fixatives differ?

A

Nuclear:
Contains glacial acetic acid
pH <4.6

Cytoplasmic:
No acetic acid
pH >4.6

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10
Q

What factors:

Retard fixation?

Enhance fixation?

A

Large size, mucus, fat, blood, cold temp

Thin slices, agitation, mild heat (37-56°C)

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11
Q

How should these be fixed?

Uterus

Brain

A

Opened/sliced thinly

Suspended whole in formalin for 2-3 weeks

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12
Q

Why and how should lung tissue be specially handled?

A

Wrap in gauze during fixation

Prevents floating due to air spaces

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13
Q

How to handle:

Mucous-rich specimens

Fatty tissues

A

Rinse with NSS before fixation

Extend fixation time or use specialized fixatives

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14
Q

What special considerations exist for preserving:

Glycogen

Iron

A

Use alcohol-based fixatives

Avoid acidic solutions that may leach metals

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15
Q

How do you prepare 1L of 10% formalin from stock?

A

100mL 37-40% formalin + 900mL distilled water
(Bonus: Buffered to pH 7 for routine use)

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16
Q

Compare formalin’s pros and cons:

A

Pros: Cheap, stable, versatile staining
Cons: Irritating fumes, dermatitis risk

(Bonus: Add 10% methanol prevents paraformaldehyde formation)

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17
Q

What makes NBF ideal for:

Iron pigments?

Elastic fibers?

A

Maintains iron in ferric state

Preserves elastin structure

(Bonus: pH 7 prevents acid hydrolysis)

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18
Q

When would you use 10% formal-saline?

A

CNS tissues

Post-mortem samples

Silver staining techniques

(Bonus: Contains 10% NaCl)

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19
Q

What are key features of formal-sublimate?

A

Contains mercuric chloride

Fixes lipids

No need for washing out

Excellent for reticulin stains

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20
Q

What specimens benefit from (Gendre’s Fixative) alcoholic formalin?

A

Sputum (preserves architecture)

Glycogen-rich tissues

(Bonus: Contains picric + acetic acid)

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21
Q

Why choose glutaraldehyde over formalin?

A

Better for EM (crosslinks proteins)

Preserves enzyme activity

Less irritating

(Bonus: Requires refrigeration)

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22
Q

When is paraformaldehyde preferred?

A

Plastic embedding

Immunohistochemistry

EM studies

(Bonus: Used at 4% concentration)

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23
Q

What are the key properties of mercuric chloride fixatives?

A

Concentration: 5-7%

Rapid penetration/hardening

Preserves cellular detail excellently

Produces black deposits (removed with iodine/alcohol)

Common in compound fixatives

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24
Q

What are the components and uses of Zenker’s solution?

A

Mercuric chloride
Potassium dichromate
Glacial acetic acid

Uses:
Small liver/spleen biopsies
Connective tissue studies
Trichrome staining

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25
How does Helly's solution differ from Zenker's?
Replaces acetic acid with formalin Best for: * Pituitary gland * Bone marrow * Blood-rich organs Preserves cytoplasmic granules
26
What makes Susa fixative unique?
Contains TCA + acetic acid + formalin No washing needed (direct to alcohol) Ideal for skin tumors Minimal shrinkage
27
When is B5 fixative specifically used?
Bone marrow biopsies Contains mercuric chloride + sodium acetate Excellent nuclear detail
28
What is the dezincification process?
Lugol's iodine (oxidizes mercury) Sodium thiosulfate (removes iodine) Running water wash
29
Which fixative would you choose for: Cytoplasmic granules Skin tumors Bone marrow
Helly's Susa B5
30
How to address: Zenker's pigments Mercury deposits
Picric acid wash Iodine-thiosulfate treatment
31
What are the key properties of chromate fixatives?
Concentration: 1-3% aqueous solutions Strong oxidizing agents (avoid reducing agents) Carcinogenic (handle with care) Excellent for carbohydrate preservation Commonly used: Potassium dichromate
32
What are the specific uses and properties of potassium dichromate?
3% aqueous solution Preserves: * Lipids and mitochondria * Cytoplasm (at pH 4.5-5.2) * Chromatin and chromosomes Component of Regaud's and Muller's fluids
33
When would you use Regaud's or Muller's fluid?
Best for: * Mitochondria studies * Mitotic figures * RBC preservation * Colloid-rich tissues (thyroid, pituitary) Contains potassium dichromate + formalin
34
What are the special applications of Orth's fluid?
Demonstrates: * Early degenerative processes * Rickettsia organisms * Myelin sheaths Contains potassium dichromate + formalin + sodium sulfate
35
What are the characteristics of lead fixatives?
4% aqueous solution Primary uses: * Acid mucopolysaccharides * Mucin preservation Warning: Forms lead carbonate precipitate over time
36
How should lead fixatives be maintained?
Storage: * Dark bottles * Acidic pH For precipitation: * Add acetic acid drops * Filter before use Avoid prolonged standing
37
Match these special stains to their optimal fixative: Mitochondria Mucins Rickettsia Carbohydrates
Regaud's fluid Lead nitrate Orth's fluid Potassium dichromate
38
How would you address: Chromate precipitates Lead carbonate formation
Wash thoroughly in running water Add acetic acid and filter
39
What are the general properties of Picric Acid Fixatives?
* Usually used in strong or saturated solutions * Chemically termed as 2,4,6-dinitrophenol * Major disadvantage: explosive when dry * Excellent for glycogen * Can be used as stain (yellow) * Small tissue fragments can be seen
40
What is Bouin's fixative used for?
* Recommended for embryos and pituitary biopsies * Excellent for preserving soft and delicate tissues * Not suitable for kidney fixation
41
What are the properties of Brasil's Alcoholic Picroformol Fixative?
* Contains TCA (trichloroacetic acid) * Better and less messy than Bouin's * Effectively fixes glycogen
42
What are the key characteristics of Glacial Acetic Acid Fixatives?
* Usually incorporated in compound fixatives * Solidifies at 17°C * Precipitates nucleoproteins and chromatin materials * Not suitable for cytoplasmic fixation
43
What is the general action of Alcohol Fixatives?
* Acts as a fixative and dehydrating agent * Excellent for glycogen * Preserves nuclear stains but dissolves fats and lipids * Rapidly denatures and precipitates proteins
44
What are the properties and uses of Methanol (100%)?
* Also known as Wood Alcohol * Used for fixing wet and dry smears, blood smears, and bone marrow biopsies * Toxic and can cause blindness
45
What are the uses of Isopropyl Alcohol (95%)?
* Also known as Rubbing Alcohol * Fixes touch preparation * Used for staining with Wright-Giemsa
46
What are the properties and applications of Ethyl Alcohol (70-100%)?
* Also known as Grain Alcohol * Simple fixative for blood, tissue films, and smears * Doesn't fix glycogen * Useful in PCR
47
What are the characteristics of Carnoy's Fixative?
* Most rapid fixative * Contains absolute alcohol, chloroform, and glacial acetic acid * Fixes and dehydrates simultaneously * Recommended for chromosomes, lymph glands, and urgent biopsies * Fixes Nissl granules (found in nerve cells) and cytoplasmic granules
48
What is Newcomer's fixative used for?
* Recommended for mucopolysaccharides and nuclear proteins * Acts as both nuclear and biochemical fixative
49
What are the main uses of Osmium Tetroxide?
* Electron microscopy * Fixes and stains fats black * Preserves mitochondria and Golgi bodies
50
What is Flemming's fixative used for?
* Nuclear structures * Permanently fixes fats * Chrome-osmium acetic acid fixative
51
What cellular components does Flemming's without Acetic Acid preserve?
* Cytoplasmic structures * Especially good for mitochondria
52
What are the properties of Trichloroacetic Acid fixative?
* Precipitates proteins * Weak decalcifying agent * Softens dense tissues
53
What is Acetone fixative best used for?
* Diffusible enzymes (phosphatases, lipases) * Brain tissues * Demonstrates Negri Bodies in Rabies * Used cold (-5 to 4°C)
54
What are the common applications of Heat Fixation?
* Frozen tissue sections * Bacteriological smears * Works by coagulating proteins
55
What are the advantages of Microwave fixation?
* Accelerates fixation and staining * Optimal temperature: 45-55°C * Speeds up decalcification
56
Which fixatives are used for Enzyme Histochemistry?
* 4% formalin * Formol saline * Acetone (for cryostat sections)
57
What are the main fixatives used for Electron Microscopy?
* Osmium Tetroxide * Glutaraldehyde * Paraformaldehyde
58
What fixatives are used for Electron Histochemistry and Immunocytochemistry?
* Karnovsky * Paraformaldehyde * Glutaraldehyde
59
What is Secondary Fixation and its purposes?
* Placing already fixed tissue in another fixative * Improves demonstration of certain substances * Makes special staining possible * Ensures complete hardening and preservation
60
What is Post-Chromatization?
* Secondary fixation using 2.5-3% potassium dichromate * Applied for one day * Acts as a mordant
61
Why and how are fixatives washed out?
* Improves staining * Removes artifacts * Tap water: removes chromates, formalin, osmic acid * 50-70% Alcohol: removes excess picric acid * Alcoholic Iodine: removes excess mercury fixatives

Histopathology Laboratory (7 decks)

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