Podcast
Questions and Answers
What is the fixative of choice for proteins?
What is the fixative of choice for proteins?
- Neutral Buffered Formalin (correct)
- Paraformaldehyde
- Osmium Tetroxide
- Glutaraldehyde
Which fixative should be avoided for nucleic acids?
Which fixative should be avoided for nucleic acids?
- Alcoholic fixatives (correct)
- HOPE
- Aldehyde fixatives
- Paraformaldehyde
What pH range should the fixative ideally be within for ultrastructure preservation?
What pH range should the fixative ideally be within for ultrastructure preservation?
- 10-14
- 1-6
- 4-9 (correct)
- 7-12
How does a hypertonic fixative solution affect cells during fixation?
How does a hypertonic fixative solution affect cells during fixation?
What is the recommended temperature for routine tissue fixation?
What is the recommended temperature for routine tissue fixation?
What is the ideal volume relationship between fixative and tissue volume?
What is the ideal volume relationship between fixative and tissue volume?
What is the main objective of tissue fixation in the laboratory?
What is the main objective of tissue fixation in the laboratory?
How do fixatives act on biological samples?
How do fixatives act on biological samples?
Why is prolonged fixation of a tissue sample a concern in immunohistochemistry?
Why is prolonged fixation of a tissue sample a concern in immunohistochemistry?
What is the role of a microtome in tissue processing?
What is the role of a microtome in tissue processing?
Which factor influences the choice of fixative and fixation protocol in a laboratory?
Which factor influences the choice of fixative and fixation protocol in a laboratory?
How does fixation contribute to tissue preparation for microscopy or analysis?
How does fixation contribute to tissue preparation for microscopy or analysis?
What is the aim of Tissue Processing?
What is the aim of Tissue Processing?
What is the last step in tissue processing before embedding?
What is the last step in tissue processing before embedding?
Why is under-fixation considered a significant problem in the fixation process?
Why is under-fixation considered a significant problem in the fixation process?
What is the main purpose of embedding medium in tissue processing?
What is the main purpose of embedding medium in tissue processing?
Which step in tissue processing involves transferring tissue through concentrated alcohol solutions?
Which step in tissue processing involves transferring tissue through concentrated alcohol solutions?
Why is over-fixation considered harmful in the fixation process?
Why is over-fixation considered harmful in the fixation process?
Frozen sections should be fixed using alcoholic fixatives.
Frozen sections should be fixed using alcoholic fixatives.
A fixative with a pH of 10 is considered ideal for tissue preservation.
A fixative with a pH of 10 is considered ideal for tissue preservation.
Hypertonic fixative solutions are preferred as they help in preventing cell shrinkage during fixation.
Hypertonic fixative solutions are preferred as they help in preventing cell shrinkage during fixation.
The temperature during fixation has no impact on the speed of the fixation process.
The temperature during fixation has no impact on the speed of the fixation process.
Using a fixative volume less than tissue volume enhances the quality of fixation.
Using a fixative volume less than tissue volume enhances the quality of fixation.
Osmium Tetroxide is recommended as a fixative for proteins.
Osmium Tetroxide is recommended as a fixative for proteins.
Dehydration in tissue processing involves replacing water with a medium that solidifies to allow thick sections to be cut.
Dehydration in tissue processing involves replacing water with a medium that solidifies to allow thick sections to be cut.
Over-fixation is considered more beneficial than under-fixation in the fixation process.
Over-fixation is considered more beneficial than under-fixation in the fixation process.
In the tissue processing step of infiltration, the tissue is placed in melted paraffin until it becomes completely infiltrated with water.
In the tissue processing step of infiltration, the tissue is placed in melted paraffin until it becomes completely infiltrated with water.
The main aim of embedding medium in tissue processing is to remove water from tissues.
The main aim of embedding medium in tissue processing is to remove water from tissues.
Fixation is a physical process that does not require any time for completion.
Fixation is a physical process that does not require any time for completion.
In the dehydration step of tissue processing, the tissue is transferred through a series of alcohol solutions of decreasing concentrations.
In the dehydration step of tissue processing, the tissue is transferred through a series of alcohol solutions of decreasing concentrations.
Formalin acts to enable intrinsic biomolecules, particularly proteolytic enzymes, in tissue samples.
Formalin acts to enable intrinsic biomolecules, particularly proteolytic enzymes, in tissue samples.
The objective of tissue fixation is to alter the cells and tissue components on a molecular level for further analysis.
The objective of tissue fixation is to alter the cells and tissue components on a molecular level for further analysis.
Fixation is usually the last step in preparing a sample of biological material for microscopy or other analysis.
Fixation is usually the last step in preparing a sample of biological material for microscopy or other analysis.
Prolonged fixation does not chemically mask targets in immunohistochemistry that antibodies bind to.
Prolonged fixation does not chemically mask targets in immunohistochemistry that antibodies bind to.
Fixatives do not have any impact on the mechanical strength or stability of cells and tissues during processing.
Fixatives do not have any impact on the mechanical strength or stability of cells and tissues during processing.
The choice of fixative and fixation protocol in a laboratory is not influenced by the planned additional processing steps and final analyses.
The choice of fixative and fixation protocol in a laboratory is not influenced by the planned additional processing steps and final analyses.
What can occur either before fixation or during any step of tissue preparation for study?
What can occur either before fixation or during any step of tissue preparation for study?
Which staining technique is used for the Leishman Stain Blood smear?
Which staining technique is used for the Leishman Stain Blood smear?
What structural abnormality might be simulated by a section showing features of autolysis artifact along with the separation of epithelium from connective tissue?
What structural abnormality might be simulated by a section showing features of autolysis artifact along with the separation of epithelium from connective tissue?
What might be seen in a histopathological image due to scoring and tearing of a tissue section?
What might be seen in a histopathological image due to scoring and tearing of a tissue section?
Which step of tissue preparation involves the mounting of a coverslip on the slide?
Which step of tissue preparation involves the mounting of a coverslip on the slide?
What might be used to avoid autolysis artifact in tissue preparation?
What might be used to avoid autolysis artifact in tissue preparation?
Which staining technique is typically used for most blood smears?
Which staining technique is typically used for most blood smears?
What is the role of Eosin in the Hematoxylin and Eosin (H&E) stain?
What is the role of Eosin in the Hematoxylin and Eosin (H&E) stain?
Which term is used for staining techniques applied when Hematoxylin and Eosin does not provide sufficient information?
Which term is used for staining techniques applied when Hematoxylin and Eosin does not provide sufficient information?
What distinguishes Eosin from Hematoxylin in staining techniques?
What distinguishes Eosin from Hematoxylin in staining techniques?
Which staining technique is commonly used as a counterstain to distinguish additional tissue features?
Which staining technique is commonly used as a counterstain to distinguish additional tissue features?
What is the typical combination of dyes used for routine staining of blood smears?
What is the typical combination of dyes used for routine staining of blood smears?
What is the primary purpose of Leishman stain in blood smear staining?
What is the primary purpose of Leishman stain in blood smear staining?
Which component of Leishman Stain primarily stains the acidic parts of the cell such as nuclei and cytoplasm of WBCs?
Which component of Leishman Stain primarily stains the acidic parts of the cell such as nuclei and cytoplasm of WBCs?
What role does Eosin play in Leishman Stain when it comes to staining blood cells?
What role does Eosin play in Leishman Stain when it comes to staining blood cells?
In Leishman Stain, how does the angle of the spreader slide affect the blood smear preparation?
In Leishman Stain, how does the angle of the spreader slide affect the blood smear preparation?
What crucial factor influences how well a blood smear is prepared using Leishman Stain?
What crucial factor influences how well a blood smear is prepared using Leishman Stain?
How does Methylene blue contribute to staining in blood smears?
How does Methylene blue contribute to staining in blood smears?
Match the staining technique with its primary purpose:
Match the staining technique with its primary purpose:
Match the staining technique with its primary role in interpretation of tissue structures:
Match the staining technique with its primary role in interpretation of tissue structures:
Match the fixative with its recommended use:
Match the fixative with its recommended use:
Match the staining technique with its common application:
Match the staining technique with its common application:
Match the fixative characteristic with its impact on tissue quality:
Match the fixative characteristic with its impact on tissue quality:
Match the staining technique with its primary purpose:
Match the staining technique with its primary purpose:
Match the staining technique with its staining components:
Match the staining technique with its staining components:
Match the fixative with its impact on tissue preservation:
Match the fixative with its impact on tissue preservation:
Match the tissue preparation step with its purpose:
Match the tissue preparation step with its purpose:
Match the staining technique component with its role in staining blood cells:
Match the staining technique component with its role in staining blood cells:
Match the tissue processing step with its effect on tissue structures:
Match the tissue processing step with its effect on tissue structures:
Match the following staining techniques with their primary usage:
Match the following staining techniques with their primary usage:
Match the following terms with their definitions:
Match the following terms with their definitions:
Match the staining techniques with their characteristics:
Match the staining techniques with their characteristics:
Match the fixative solutions with their effects on tissues:
Match the fixative solutions with their effects on tissues:
Match the tissue preparation steps with their descriptions:
Match the tissue preparation steps with their descriptions:
Match the fixative pH levels with their impact on tissues:
Match the fixative pH levels with their impact on tissues:
Flashcards
Tissue Fixation
Tissue Fixation
Preserving cells and tissue components close to their normal state for microscopic preparation.
Fixative pH
Fixative pH
Should be in the physiological range (4-9) for general preservation, buffered between 7.2-7.4 for ultrastructure.
Fixative Osmolarity
Fixative Osmolarity
Maintain the same concentration as cells to prevent shrinkage or swelling during fixation.
Tissue Size for Fixation
Tissue Size for Fixation
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Fixative Volume
Fixative Volume
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Tissue Processing
Tissue Processing
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Dehydration
Dehydration
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Clearing
Clearing
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Infiltration/Impregnation
Infiltration/Impregnation
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Embedding
Embedding
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H&E Stain
H&E Stain
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Artifacts
Artifacts
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Study Notes
Tissue Fixation
- Objective of tissue fixation is to preserve cells and tissue components, and keep them as close to normal as possible, allowing for the preparation of thin and stained sections.
- Fixation is usually the first step in a process to prepare a sample of biological material for microscopy or other analysis.
Factors Affecting Fixation
- pH of the fixative: Should be kept in the physiological range, between pH 4 to 9; pH for ultrastructure preservation should be buffered between 7.2 to 7.4.
- Osmolarity of the fixative: Avoid hypertonic solutions (cause cell shrinkage) and hypotonic solutions (result in cell swelling and poor fixation).
- Size of the specimen: Ideal thickness is 1-4mm.
- Volume of the fixative: At least 15-20 times greater than tissue volume.
- Temperature: High temperature increases the speed of fixation, but care is required to avoid cooking the specimen; fixation is routinely carried out at room temperature.
- Duration: As a general rule, 1 hour per 1mm.
Types of Fixation
- Neutral Buffered Formalin and Paraformaldehyde are the fixatives of choice for proteins.
- Frozen Sections and Chemical Fixatives are used for enzymes.
- Alcoholic fixatives and HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) are used for nucleic acids.
- Osmium Tetroxide and Glutaraldehyde/Osmium Tetroxide are used for lipids.
- Frozen Sections and Chemical fixatives are used for mucopolysaccharides.
- Bouin Solution and Neutral Buffered Formalin are used for biogenic amines and glycogen.
Tissue Processing
- The aim of tissue processing is to remove water from tissues and replace it with a medium that solidifies, allowing for the preparation of thin sections.
- Three steps involved in tissue processing: Dehydration, Clearing, and Infiltration and impregnation.
Dehydration
- The tissue is transferred through a series of increasingly concentrated alcohol solutions, ending in 100%, which removes all water.
Clearing
- The ethanol is replaced by an organic solvent miscible with both alcohol and the embedding medium, giving the tissue a translucent appearance.
Infiltration and Impregnation
- The tissue is placed in melted paraffin until it becomes completely infiltrated with this substance.
Embedding
- The embedding medium has three crucial functions: to give support to the tissue, to prevent distortion of the tissue during cutting, and to aid in staining.
Staining
- The term "routine staining" refers to the Hematoxylin and Eosin stain (H&E) that is routinely used with all tissue specimens to reveal the underlying tissue structures and conditions.
- Leishman stain is used for blood smears, and is characterized by shades of blue, pink, and purple, giving a clear differentiation and understanding of cellular morphology.
H&E Stain
- Hematoxylin stains DNA in the cell nucleus, RNA-rich portions of the cytoplasm, producing a dark blue or purple color.
- Eosin stains other cytoplasmic structures and collagen, producing a pink color.
Artifacts
- An artifact is an artificial structure or tissue alteration on a prepared microscopic slide, caused by an extraneous factor.
- Artifacts can occur either before fixation or during any of the steps involved in the preparation of tissue for study.
- Examples of artifacts include improper prefixation, microtomy, and staining.
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Description
Test your knowledge on tissue fixation methods including the types of fixatives commonly used to stabilize proteins, nucleic acids, and microsubstances in tissues. Identify the target fixative of choice and which fixatives to avoid for different tissue components.
