71 Questions
What is the fixative of choice for proteins?
Neutral Buffered Formalin
Which fixative should be avoided for nucleic acids?
Alcoholic fixatives
What pH range should the fixative ideally be within for ultrastructure preservation?
4-9
How does a hypertonic fixative solution affect cells during fixation?
Causes cell shrinkage
What is the recommended temperature for routine tissue fixation?
Room temperature
What is the ideal volume relationship between fixative and tissue volume?
At least 20 times greater fixative volume than tissue volume
What is the main objective of tissue fixation in the laboratory?
To preserve cells and tissue components as close to normal as possible
How do fixatives act on biological samples?
By altering cells or tissues at a molecular level
Why is prolonged fixation of a tissue sample a concern in immunohistochemistry?
It chemically masks the protein targets preventing antibody binding
What is the role of a microtome in tissue processing?
Cutting the specimen into super thin slices
Which factor influences the choice of fixative and fixation protocol in a laboratory?
The additional processing steps and final analyses planned
How does fixation contribute to tissue preparation for microscopy or analysis?
By preserving cells and tissues close to their normal state
What is the aim of Tissue Processing?
To remove water from tissues and replace with a medium that solidifies
What is the last step in tissue processing before embedding?
Infiltration
Why is under-fixation considered a significant problem in the fixation process?
It can result in inappropriate assay results
What is the main purpose of embedding medium in tissue processing?
To support and prevent distortion during cutting
Which step in tissue processing involves transferring tissue through concentrated alcohol solutions?
Dehydration
Why is over-fixation considered harmful in the fixation process?
It can cause damage to the tissue structure
Frozen sections should be fixed using alcoholic fixatives.
True
A fixative with a pH of 10 is considered ideal for tissue preservation.
False
Hypertonic fixative solutions are preferred as they help in preventing cell shrinkage during fixation.
False
The temperature during fixation has no impact on the speed of the fixation process.
False
Using a fixative volume less than tissue volume enhances the quality of fixation.
False
Osmium Tetroxide is recommended as a fixative for proteins.
False
Dehydration in tissue processing involves replacing water with a medium that solidifies to allow thick sections to be cut.
False
Over-fixation is considered more beneficial than under-fixation in the fixation process.
False
In the tissue processing step of infiltration, the tissue is placed in melted paraffin until it becomes completely infiltrated with water.
False
The main aim of embedding medium in tissue processing is to remove water from tissues.
False
Fixation is a physical process that does not require any time for completion.
False
In the dehydration step of tissue processing, the tissue is transferred through a series of alcohol solutions of decreasing concentrations.
False
Formalin acts to enable intrinsic biomolecules, particularly proteolytic enzymes, in tissue samples.
False
The objective of tissue fixation is to alter the cells and tissue components on a molecular level for further analysis.
False
Fixation is usually the last step in preparing a sample of biological material for microscopy or other analysis.
False
Prolonged fixation does not chemically mask targets in immunohistochemistry that antibodies bind to.
False
Fixatives do not have any impact on the mechanical strength or stability of cells and tissues during processing.
False
The choice of fixative and fixation protocol in a laboratory is not influenced by the planned additional processing steps and final analyses.
False
What can occur either before fixation or during any step of tissue preparation for study?
Artifact
Which staining technique is used for the Leishman Stain Blood smear?
Wright-Giemsa stain
What structural abnormality might be simulated by a section showing features of autolysis artifact along with the separation of epithelium from connective tissue?
Pemphigus
What might be seen in a histopathological image due to scoring and tearing of a tissue section?
Nick in knife edge
Which step of tissue preparation involves the mounting of a coverslip on the slide?
Finalization
What might be used to avoid autolysis artifact in tissue preparation?
Fixative solutions like formalin
Which staining technique is typically used for most blood smears?
Wright-Giemsa stain
What is the role of Eosin in the Hematoxylin and Eosin (H&E) stain?
Stains collagen pink
Which term is used for staining techniques applied when Hematoxylin and Eosin does not provide sufficient information?
Special stains
What distinguishes Eosin from Hematoxylin in staining techniques?
Stains collagen pink
Which staining technique is commonly used as a counterstain to distinguish additional tissue features?
Eosin stain
What is the typical combination of dyes used for routine staining of blood smears?
Acidic (eosin) and basic (methylene blue)
What is the primary purpose of Leishman stain in blood smear staining?
To highlight the different blood cell components in shades of blue and pink
Which component of Leishman Stain primarily stains the acidic parts of the cell such as nuclei and cytoplasm of WBCs?
Methylene blue
What role does Eosin play in Leishman Stain when it comes to staining blood cells?
Stains basic parts of cells like eosinophilic granules
In Leishman Stain, how does the angle of the spreader slide affect the blood smear preparation?
Affects the length of the smear produced
What crucial factor influences how well a blood smear is prepared using Leishman Stain?
The speed at which the spreader slide is moved
How does Methylene blue contribute to staining in blood smears?
Highlights Hb in red cells
Match the staining technique with its primary purpose:
Hematoxylin and Eosin (H&E) stain = Highlighting tissue structures Leishman Stain = Staining blood smears Methylene blue stain = Contributing to staining in blood smears Eosin stain = Staining acidic parts of the cell like nuclei and cytoplasm
Match the staining technique with its primary role in interpretation of tissue structures:
Hematoxylin and Eosin (H&E) stain = Distinguishing additional tissue features as a counterstain Leishman Stain = Affecting blood smear preparation with the angle of the spreader slide Methylene blue stain = Providing information when H&E does not suffice Eosin stain = Staining WBCs and acidic cell parts in Leishman Stain
Match the fixative with its recommended use:
Osmium Tetroxide = Recommended for proteins Alcoholic fixatives = Should be used for frozen sections Fixatives with pH of 10 = Considered ideal for tissue preservation Fixatives to avoid for nucleic acids = Not recommended for certain types of samples
Match the staining technique with its common application:
Hematoxylin and Eosin (H&E) stain = Routine staining of blood smears Leishman Stain = Primary stain used for blood smears Methylene blue stain = Typically used for most blood smears Eosin stain = Role in H&E for staining tissue structures
Match the fixative characteristic with its impact on tissue quality:
Using fixative volume less than tissue volume = Enhances fixation quality Prolonged fixation time = Concern in immunohistochemistry Over-fixation = Considered harmful in fixation process Under-fixation = Significant problem in fixation quality
Match the staining technique with its primary purpose:
Hematoxylin and Eosin (H&E) stain = Providing contrast to tissue structures Leishman stain = Staining acidic parts of cells such as nuclei and cytoplasm of WBCs Eosin stain = Counterstaining to distinguish additional tissue features Methylene blue stain = Highlighting specific features of interest in tissue
Match the staining technique with its staining components:
Hematoxylin and Eosin (H&E) stain = Hematoxylin and Eosin Leishman stain = Methylene blue and Eosin Eosin stain = Eosin only Methylene blue stain = Methylene blue only
Match the fixative with its impact on tissue preservation:
Formalin = Enabling intrinsic biomolecules in tissue samples Osmium Tetroxide = Recommended for proteins Alcoholic fixatives = Recommended for frozen sections Hypertonic fixative solutions = Preventing cell shrinkage during fixation
Match the tissue preparation step with its purpose:
Infiltration = Replacing water with embedding medium in tissues Embedding = Solidifying medium to allow thick sections to be cut Dehydration = Replacing water with alcohol solutions of decreasing concentrations Mounting on slides = Drying sections on a hot plate or hot air oven
Match the staining technique component with its role in staining blood cells:
Hematoxylin = Staining cell nuclei Eosin = Staining cytoplasm of cells Methylene blue = Contributing to overall contrast in blood smears Leishman Stain components = Staining acidic parts of the cell
Match the tissue processing step with its effect on tissue structures:
Sectioning with a diamond knife in an ultra microtome = Cutting 0.1-0.5 µm thick tissue sections on copper grids Paraffin sectioning using a Microtome = Mounting sections onto glass slides and labeling them Staining sections with appropriate dyes = Adding contrast to tissues and highlighting specific features Storing paraffin-embedded sections at specific temperatures = Ensuring long-term preservation of tissue samples
Match the following staining techniques with their primary usage:
Leishman Stain Blood smear stained with Wright-Giemsa stain = Differentiate various blood cell types Hematoxylin and Eosin = Visualize different tissue structures Hematoxylin only = Highlight cell nuclei Eosin only = Staining cytoplasmic components
Match the following terms with their definitions:
Artifact = Artificial structure on a microscopic slide due to external factors Autolysis artifact = Features seen in tissue sections where fixation was improper Microtomy artifact = Scoring and tearing of tissue sections during cutting Embedding medium = Substance used to support tissues during sectioning
Match the staining techniques with their characteristics:
Hematoxylin and Eosin = Two dyes used to stain nuclei and cytoplasm respectively Leishman Stain Blood smear = Primarily stains acidic parts of cells like WBC nuclei Methylene blue = Contributes to staining blood smears by highlighting certain cell components Eosin in Leishman Stain = Aids in staining cytoplasmic components of blood cells
Match the fixative solutions with their effects on tissues:
Normal saline = Does not properly fix tissue leading to autolysis features Formalin = Acts on intrinsic biomolecules in tissue samples Hypertonic fixative solutions = Prevents cell shrinkage during fixation Osmium Tetroxide = Recommended for fixing proteins in tissues
Match the tissue preparation steps with their descriptions:
Infiltration = Tissue placed in melted paraffin for complete infiltration Dehydration = Replacing water with a solidifying medium for sectioning Mounting of coverslip = Final step involving covering the tissue section on a slide Microtomy = Process involving cutting of tissue sections for microscopy
Match the fixative pH levels with their impact on tissues:
pH 10 fixative = Considered ideal for tissue preservation Hypertonic fixatives = Preferred to prevent cell shrinkage during fixation Neutral pH fixatives = Used to preserve tissue ultrastructure Acidic fixatives = Not recommended for nucleic acids preservation
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.
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.
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