Ch. 1 Methods Flashcards
List the 10 levels of organismal organization.
1) Whole organism
2) Organ system
3) Organs
4) Tissues
5) Cells
6) Organlles
7) Supramolecular assemblies
8) Macromolecules
9) Building block molecules
10) Atoms/ions
List the 4 anatomical sciences.
1) Gross anatomy
2) Histology
3) Embryology
4) Neuroanatomy
The scientific study of microscopic structures of tissues and organs of the body.
What is histology?
What is another name for histology?
Microscopic anatomy (microanatomy)
Today, students in histology laboratories use either {…} or {…} microscopes.
1) Light
2) Virtual
In the past, a more detailed interpretation of microscopic structures was done with the {…} microscope (EM).
Electron
What were the 2 types of electron microscopes used in the past for histology?
1) Transmission electron microscope (TEM)
2) Scanning electron microscope (SEM)
Now, the {…} (AFM) and a variety of {…} microscopic techniques can provide images that are comparable or higher (e.g., AFM) in resolution to those obtained with the TEM.
NOTE: 1st blank is 3 words
1) Atomic force microscopy
2) Super-resolution
What are the 3 steps for tissue preparation?
1) Formalin fixation
2) Paraffin embedding
3) Hemotoxylin & eosin (H&E) staining
The use of a chemical or chemical mixture to permanently preserve the tissue structure for subsequent treatments.
What is fixation?
When should tissue specimens be fixed?
As soon as they are removed from the body
What are the 4 reasons for sample fixation?
1) Terminate cell metabolism
2) Prevent enzymatic degradation
3) Kill pathogenic microbes
4) Harden tissue
This commonly used fixative is a 37% aqueous solution of formaldehyde.
What is formalin?
What is the purpose of embedding a specimen in paraffin?
To permit sectioning (i.e., allowing the specimen to be thinly sliced)
What are the 4 steps of paraffin embedding?
1) Washing specimen after fixation
2) Dehydrating specimen in series of alcohols
3) Clearing out the alcohol using organic solvents
4) Infiltration in melted paraffin
The specially designed slicing machine used to slice the hardened paraffin blocks into sections for slide mounting.
What is a microtome?
A solution that hardens into a permanent mount that keeps the specimen attached to the glass and prevents deterioration of the specimen over time.
What is a mounting medium?
Why must paraffin sections be washed in organic solvents before staining?
Paraffin sections are colorless, thus making them unsuitable for light microscopic examination
What are the 5 steps to staining a tissue section?
1) Dissolve the paraffin w/ organic solvents
2) Rehydrate slide via a series of alcohol solutions in descending concentration
3) Stain w/ hematoxylin in water
4) Dehydrate slide with alcohol solutions in ascending order
5) Stain with eosin in alcohol
Under what 2 conditions might a surgeon request a frozen section?
1) No pre-op diagnosis available
2) Unexpected intraoperative findings
What are the 3 main steps in frozen-section preparation?
1) Freezing the sample tissue
2) Sectioning the frozen tissue
3) Staining the cut sections
What are the 3 most commonly used stains for frozen sections?
1) Hematoxylin & eosin (H&E)
2) Methylene blue
3) Periodic acid-Schiff (PAS)
What are the 3 facets on which histochemical and cytochemical procedures are based?
1) Specific binding of a dye
2) Use of fluorescent dye-labeled antibody of a particular cell component
3) Inherent enzymatic activity of cell component
A process used to localize large molecules present in cells by incorporating radiolabeled precursors into cells and tissues before fixation.
What is autoradiography?
List 4 macromolecular complexes found within tissue sections.
1) Nucleoproteins
2) Intracellular cytoskeletal proteins
3) Extracellular proteins
4) Membrane phospholipid-protein (or carbohydrate) complexes
List 3 large molecules that are lost during the routine preparation of H&E-stained (i.e., aqueous) sections.
1) Glycogen
2) Proteoglycans
3) Glycosaminoglycans
An {…} dye, such as {…}, carries a net negative charge on its colored portion and is described by the general formula [Na+ dye-].
1) Acidic
2) Eosin
A {…} dye carries a net positive charge on its colored portion and is described by the general formula [dye+ Cl-].
2) Basic
True or False: Hematoxylin is a basic dye.
False; hematoxylin does not meet the definition of a strict basic dye but has properties that closely resemble those of a basic dye
True or False: The color of a dye is not related to whether it is basic or acidic.
True
What 3 functional groups make up the anionic components of cells and tissue?
1) Phosphate groups (nucleic acids)
2) Sulfate groups (glycosaminoglycans)
3) Carboxylate groups (proteins)
The ability of a sample’s anionic components to react with a basic dye.
HINT: [G., base-loving]
What is basophilia?
A fixative that acts as an intermediate link between the tissue component and a dye (i.e., hematoxylin), allowing said hematoxylin, which isn’t strictly basic, to resemble a basic dye.
What is a mordant?
The reaction of cationic groups with an acidic dye.
HINT: [Gr., acid-loving]
What is acidophilia?
Acidic dyes react with the cationic groups in cells and tissues, particularly with the ionized {…} groups of proteins.
Amino
Certain basic dyes react with tissue components that shift their normal color from blue to red or purple; this absorbance change is called {…}.
Metachromasia
The underlying mechanism for metachromasia is the presence of {…} within the tissue.
Polyanions
What is the fundamental basis of the periodic acid-Schiff and Feulgen reactions?
The ability of bleached basic fuchsin (Schiff reagent) to react with aldehyde groups, resulting in a distinctive red color
The periodic acid-Schiff (PAS) reaction stains {…} and {…} macromolecules.
HINT: 2nd blank is a hyphenate compound adjective
1) Carbohydrates
2) Carbohydrate-rich
Feulgen staining, which also uses the Schiff reagent, relies on mild hydrochloric acid hydrolysis to stain this macromolecule.
What is DNA?
Which components of a DNA molecule are being cleaved during the acid hydrolysis of the Feulgen reaction?
Purines are being cleaved from the deoxyribose sugar
Following the hydrolytic cleaving of purines from deoxyribose sugar in a Feulgen reaction, what happens to the sugar ring?
The sugar ring opens with the formation of aldehyde groups that then react with the Schiff reagent to produce the red/magenta color
The reaction of the Schiff reagent with DNA is {…}, meaning that the product of this reaction is measurable and proportional to the amount of DNA.
Stoichiometric
How can the stoichiometric aspect of the Feulgen reaction be quantitatively applied in a lab setting?
The stoichiometry makes the reaction ideal for using spectrophotometric methods to quantify the amount of DNA in the nucleus
What is the resolving power of the human eye?
0.2 mm
The ability of a lens or optical system to produce several images of closely positioned objects.
What is resolving power?
What is the resolving power of a bright-field microscope?
~0.2 µm (micrometers)
What is the most abundant extracellular cation?
Sodium
What is the most abundant intracellular cation?
Potassium
What is the most common type of microscope used by students and researchers?
Bright-field (light) microscope
List the 5 major parts of the bright-field microscope.
1) Light source
2) Condenser lens
3) Stage
4) Objective lens
5) Ocular lens
Organs are three-dimensional, whereas histologic sections are only…
Two-dimensional
A defect caused by an error in the tissue preparation process.
What is an artifact?
When do artifacts occur during tissue preparation?
They occur during all stages of tissue preparation
The phase contrast microscope enables the examination of {…} cells and tissues, which is especially useful for {…} cells.
1) Unstained
2) Living
How is a phase contrast microscope capable of being used to examine unstained specimens?
It takes advantage of the small differences in refractive index of different parts of a cell or tissue sample
What are the two modifications of the phase contrast microscope, and what are their functions?
1) Interference - quantifies tissue mass
2) Differential inference - assesses surface properties of cells & other biological objects
In this type of microscopy, no direct light from the light source is gathered by the objective lens. Only light that has been scattered or diffracted by structures in the specimen reaches the objective.
What is dark-field microscopy?
The minimum point-to-point distance between two distinguishable details.
What is resolution?
Any microscopy technique that increases resolution of a conventional light microscope dictated by the diffraction barrier by at least a factor of 2.
What is super-resolution microscopy?
The advent of super-resolution microscopy has increased optical resolution from 0.2 µm to…
About 10 nm
How does the transmission electron microscope (TEM) produce an image, and how does this process generally differ from how a light microscope produces an image?
A beam of electrons interacts with the specimen rather than a beam of light like a light microscope uses
