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Questions and Answers
What is the primary role of restriction enzymes in prokaryotes?
What is the primary role of restriction enzymes in prokaryotes?
Which component of lysis buffers is responsible for destabilizing the cell membrane?
Which component of lysis buffers is responsible for destabilizing the cell membrane?
What is the role of isopropyl alcohol in DNA extraction?
What is the role of isopropyl alcohol in DNA extraction?
Which enzyme would be used to target and degrade the peptidoglycan layer of bacterial cell walls?
Which enzyme would be used to target and degrade the peptidoglycan layer of bacterial cell walls?
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Why is a methyl group significant in the context of DNA protection from restriction enzymes?
Why is a methyl group significant in the context of DNA protection from restriction enzymes?
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Which type of microscope is specifically used in tissue culture?
Which type of microscope is specifically used in tissue culture?
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What is the purpose of fixation in tissue preparation?
What is the purpose of fixation in tissue preparation?
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Which fixative is commonly used to treat tissue samples?
Which fixative is commonly used to treat tissue samples?
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How thick are the sections typically cut using a microtome?
How thick are the sections typically cut using a microtome?
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Which microscope uses an invisible beam for imaging?
Which microscope uses an invisible beam for imaging?
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What is the purpose of staining sections during tissue preparation?
What is the purpose of staining sections during tissue preparation?
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What is the freezing technique used for in tissue preparation?
What is the freezing technique used for in tissue preparation?
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What are components stained with hematoxylin referred to as?
What are components stained with hematoxylin referred to as?
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What property of eosin allows it to stain cytoplasmic components pink?
What property of eosin allows it to stain cytoplasmic components pink?
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Which dye is used for the visualization of DNA during electrophoresis?
Which dye is used for the visualization of DNA during electrophoresis?
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What is the purpose of adding glycerol to the sample during protein electrophoresis?
What is the purpose of adding glycerol to the sample during protein electrophoresis?
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What is the primary function of sodium dodecyl sulfate (SDS) in protein electrophoresis?
What is the primary function of sodium dodecyl sulfate (SDS) in protein electrophoresis?
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What type of cells are characterized as the most primitive?
What type of cells are characterized as the most primitive?
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What is the role of bromphenol blue in the electrophoresis process?
What is the role of bromphenol blue in the electrophoresis process?
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What happens to smaller molecules during electrophoresis through a polyacrylamide gel?
What happens to smaller molecules during electrophoresis through a polyacrylamide gel?
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Which component is primarily responsible for staining areas of the cytoplasm that have an acidic pH?
Which component is primarily responsible for staining areas of the cytoplasm that have an acidic pH?
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Study Notes
Types of Microscopes
- Microscopes using visible light:
- Optical light microscope
- Modified microscopes
- Phase contrast microscope (used in tissue culture)
- Interference microscope
- Polarizing microscope
- Dark field microscope
- Dissecting microscope (stereomicroscope) (used in surgery)
Microscopes using invisible beams:
- Ultraviolet microscope
- X-ray microscope
- Electron microscope (two types):
- Transmission electron microscope
- Scanning electron microscope
Tissue Sampling
- A small piece of tissue (biopsy) is obtained under anesthesia or after death (autopsy).
Fixation
- Fixation treats tissue with chemicals or physical agents to prevent autolysis (enzyme digestion).
- Samples should be treated with fixatives (e.g., formaldehyde, glutaraldehyde) as soon as possible after removal.
Dehydration
- Tissues are dehydrated using ascending concentrations of alcohol to prevent rapid shrinkage.
Sectioning
- Paraffin blocks are placed in a microtome.
- The microtome cuts paraffin blocks into thin tissue-containing slices.
- Slices are placed on glass slides.
- Paraffin slice thickness ranges from 3-5 μm.
Freezing Technique
- Tissues are frozen using liquid nitrogen.
- Sections are cut inside a cold cabinet using a cryostat.
- Sections are then stained and examined.
Hematoxylin Dye
- Hematoxylin is a basic dye that stains acidic cell components bluish.
- Examples of acidic components include the nucleus (DNA, RNA), and regions of the cytoplasm rich in ribosomes.
Basophilic Components
- Cell components stained with Hematoxylin are termed basophilic.
Eosin Dye
- Eosin is an acid that stains basic cell components pinkish.
- Most cytoplasmic components are basic, so they stain pink.
Acidophilic Components
- Cytoplasmic elements stained with eosin are termed acidophilic.
Staining Results for Hematoxylin and Eosin
- Hematoxylin stains the nucleus and acidic regions of the cytoplasm blue
- Eosin stains basic regions of the cytoplasm pink and collagen fibers.
Protein Denaturation
- Reducing disulfide linkages overcomes tertiary and quaternary protein structures.
Polyacrylamide Gel
- Polymerized acrylamide forms a mesh-like matrix suitable for separating proteins.
Other Staining Components
- Bromophenol blue: tracking dye used to visualize samples
- Glycerol: increases sample density to ensure the sample falls to the bottom of the well
- Staining is done overnight with agitation to help ensure uniformity.
Prokaryotic vs Eukaryotic Cells
- Prokaryotic cells lack a definite nucleus and DNA floats in the cytoplasm, protected by restriction enzymes.
- Restriction enzymes cut foreign DNA (like viruses) into small pieces.
- DNA in a prokaryotic cell is not cut by these enzymes due to a methyl group attached to it.
- Eukaryotic cells have a definite nucleus and have DNAse enzymes that cut DNA.
Cell Lysis Methods
- EDTA: eliminates divalent cations to destabilize cell components.
- EDTA inhibits DNase activity—it chelates the magnesium required for DNase activity.
- Lysozyme: digests the peptidoglycan layer.
Additional Enzymes for Cell Lysis
- Cellulase for plant cells
- Lyticase for yeast cells
- Proteinase K for animal cells
DNA Precipitation
- DNA precipitation uses isopropyl or ethanol to ppt the DNA along with monovalent cations (Na+ and K+).
- Dehydration by the alcohol precipitates the DNA.
Gel Electrophoresis
- A technique in molecular biology to separate DNA and RNA by charge and/or size.
Charge of Biological Molecules
- Many biological molecules have both positive and negative charges.
- The sum of these charges determines the overall charge.
- At neutral pH, proteins have a unique charge and DNA and RNA bases are negatively charged.
Agarose Gel Electrophoresis
- A porous material derived from red seaweed.
- Acts as a sieve for separating DNA fragments.
- Smaller fragments travel faster than large fragments.
- Lower concentration gels have larger pores, which resolves larger fragments better.
Ethidium Bromide
- Visualizes DNA sample bands.
- Intercalates/inserts into the nitrogenous bases of DNA.
- When exposed to UV light, it fluoresces, allowing visualization of the DNA.
Loading Dye
- Contains one or two tracking dyes that migrate through the gel.
- Allows monitoring of electrophoresis progress.
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Description
Explore the various types of microscopes, including those that utilize visible and invisible light, as well as techniques for tissue sampling, fixation, dehydration, and sectioning. This quiz will enhance your understanding of microscopy and histology processes essential in biological sciences.
