Histology Tissue Preparation

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Questions and Answers

What is the primary purpose of tissue fixation?

To preserve the morphologic and chemical integrity of cells in a life-like manner (correct)

To harden tissues for light microscopy

To remove fat from tissue samples

To prepare tissues for electron microscopy

What is the dimensional range for tissue samples in electron microscopy?

5mm2 x 1cm

1-2mm2 (correct)

2cm2 x 0.4cm

10cm2 x 2cm

What is the first and most critical step in tissue processing?

Embedding

Sectioning

Dehydration

Tissue fixation (correct)

What is the recommended treatment for tissue samples with mucus?

Wash with NSS Signup and view all the answers

What is the dimensional range for tissue samples in light microscopy?

2cm2 x 0.4cm Signup and view all the answers

What is the recommended treatment for tissue samples with fat?

Cut into thin slices and fix Signup and view all the answers

What is the optimal temperature range for a fixative?

Room temp to 45ºC Signup and view all the answers

What is the result of hypertonicity on cells during fixation?

Cell shrinkage Signup and view all the answers

What is a characteristic of a good fixative that prevents further processing?

Makes cellular contents resistant to further processing Signup and view all the answers

What is the temperature range for microwave processing?

Up to 65ºC Signup and view all the answers

What is the purpose of a fixative in tissue processing?

To preserve the tissue for further analysis Signup and view all the answers

What is the temperature range for electron microscopy?

0 - 4ºC Signup and view all the answers

What is the byproduct of methanol oxidation?

Gas produced by oxidation of methanol Signup and view all the answers

Which type of fixative is best for iron-containing pigments and elastic fibers?

F.METALLIC FIXATIVES Signup and view all the answers

What is the disadvantage of using Formaldehyde as a fixative?

It is inert to phospholipids and neutral fats Signup and view all the answers

How long does it take to prepare a tissue using Formaldehyde?

4 to 24 hours Signup and view all the answers

What is the advantage of using Formol-Corrosive (Formol Mercuric Chloride) as a fixative?

It does not need washing Signup and view all the answers

What is the disadvantage of using Formol-Corrosive (Formol Mercuric Chloride) as a fixative?

It forms mercuric chloride deposits Signup and view all the answers

What type of tissue is Formol-Corrosive recommended for?

Routine post-mortem tissue Signup and view all the answers

What is the percentage of Mercuric chloride in the most common metallic fixative?

5-7% Signup and view all the answers

What is the primary advantage of using Phloroglucinol-Nitric Acid as a decalcifying agent?

It is the fastest decalcifying agent Signup and view all the answers

What is the purpose of adding urea or Sodium Thiosulfate/sulfate to a decalcifying agent?

To render the best and accurate result Signup and view all the answers

What is the composition of ROH (DHD)?

70% ROH (DHD) is an alkyl alcohol Signup and view all the answers

What is the function of Formol-Nitric Acid as a decalcifying agent?

It acts as a tissue softener Signup and view all the answers

What is a characteristic of a good decalcifying agent?

It is rapid and inexpensive Signup and view all the answers

What is a common brand of decalcifying agent that contains Na2EDTA?

Versene Signup and view all the answers

What is the composition of Perenyi's Fluid?

Nitric acid + chromic acid + ROH Signup and view all the answers

What is the primary purpose of a decalcifying agent?

To remove calcium ions from tissues Signup and view all the answers

What is the main purpose of performing clearing in tissue processing?

To create a bridge between the process of dehydration and infiltration Signup and view all the answers

Which of the following characteristics is NOT a desirable feature of a good clearing agent?

It should produce tissue shrinkage Signup and view all the answers

Which clearing agent is typically used for tough tissues and large specimens?

Chloroform Signup and view all the answers

What is a potential risk associated with prolonged exposure to clearing agents?

Respiratory problems Signup and view all the answers

Why is cedarwood oil used for smooth muscle and CNS tissues?

It requires deeper penetration Signup and view all the answers

What is a characteristic of aniline oil?

It is slow and expensive to use Signup and view all the answers

Why is chloroform not used for delicate tissues?

It is toxic to the liver Signup and view all the answers

What is a common problem associated with excessive clearing?

Tissue brittleness Signup and view all the answers

Study Notes

Fixation

Larger tissues require longer fixation time
Fixation is the killing, penetration, and hardening of tissues
Light Microscopy: 2cm2 x 0.4cm, Electron Microscopy: 1-2mm2
Primary purpose: Preserve the morphologic and chemical integrity of the cell in a life-like manner as possible
Characteristics of a good fixative:
- Cheap
- Stable
- Safe
- Quick
- Inhibits bacterial decomposition
- Produces minimum shrinkage
- Rapid and even penetration
- Hardens the tissue
- Makes cellular contents resistant to further processing
No single fixative has all the mentioned characteristics

Temperature for Fixation

Room temperature to 45°C: Optimal temperature (routine)
40°C: Tissue processors
Up to 65°C: Microwave processing
0 - 4°C: Electron microscopy
100°C: Tuberculosis
60°C: Rapid biopsy

Osmolality

Hypertonicity: Cell shrinkage
Isotonicity and Hypotonicity: Cell swelling
Maintain tissues at slightly hypertonic solution

Metallic Fixatives

Mercuric Chloride: Most common metallic fixative; 5-7%
Zenker: For iron-containing pigments and elastic fibers
Zenker-Formol (Helly’s): Carnoy-Lebron, Heidenhain’s Susa, B5

Formol-Corrosive (Formol Mercuric Chloride)

Saturated aq. Mercuric chloride + 40% Formaldehyde
Recommended for routine post-mortem tissues and Silver Reticulum staining methods
Advantage: Does not need washing, fixes lipids
Disadvantage: Forms mercuric chloride deposits

Characteristics of a Good Decalcifying Agent

Do not cause cell destruction
Rapid, cheap, and inexpensive
Safe
Readily available
Should also render best and accurate result

Decalcifying Agents

Nitric Acid Solution (10%): Most commonly used
Formol-Nitric Acid
Perenyi’s Fluid: Acts as tissue softener
Phloroglucinol-Nitric Acid: Fastest agent

Clearing Agents

Chloroform (6-24 hours): Tough tissues and large specimens
Cedarwood Oil (2-3 days): For smooth muscle, CNS (requires deeper penetration)
Aniline Oil: For insects, embryos, and delicate tissues
Clove Oil: Minimum shrinkage of tissues
Carbon Tetrachloride: Tough tissues and large specimens

Characteristics of Good Clearing Agent

Miscible with alcohol
Miscible with paraffin wax
Does not produce tissue shrinkage
Makes tissue transparent
Does not dissolve aniline dyes

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Description

Understanding the process of tissue preparation for microscopy, including fixation and hardening of tissues. Learn about the importance of this critical step in histology.