MCDB 428 Exam 1 Questions with Verified Solutions Latest 2025
Normal tissue culture cell size - Answers Normally 20-30 µm in diameter
Size of most eukaryotic cells - Answers 5 -100 µm
Kinds of light microscopy - Answers Transmission
Fluorescence
Transmission microscopy - Answers White light that has passed through the sample is
collected at the eyepiece
Epifluorescence microscopy - Answers Specimen is illuminated with the light of a specific
wavelength. This light is reabsorbed and only re-emitted fluorescence of a specific wavelength
is collected at the eyepiece.
Contrast - Answers Major challenge in transmission microscopic techniques
Because individual cells are predominantly transparent at high levels of magnification
Bright field - Answers Light transmitted straight through the specimen
Phase contrast - Answers Phase alterations of light transmitted through the specimen are
translated into brightness changes
Can see details
DIC - Answers Highlights edges where there is a steep change of refractive index
Different wavelengths
Dark field - Answers Specimen lit from side and only scattered light is seen
Used to look at fluorescence
Staining of cell components - Answers Because you can't see details by just microscopy alone
Paraffin section of urine-collecting ducts are stained with H&E
,Hematoxylin - Answers Basic dye that stains the nucleus
Eoisin - Answers Acidic dye that stains the cytosol
Fluorescence microscopy - Answers First barrier filers - only allows blue light through from light
source
Beam-splitting mirror - reflects light below 510 nm but transmits light above it
Second barrier filter - passes green light only to eyepiece
Multi-probe fluorescence microscopy - Answers Often antibodies are used to recognize a
protein, located on a particular structure, followed by a fluorescent second antibody
Can reveal subcellular compartments and cell dynamics
Primary antibody - Answers Rabbit antibody directed against antigen
Not usually labeled
Secondary antibody - Answers Marker-coupled antibody directed against rabbit antibodies
Kinds of tags that can be coupled to secondary antibodies - Answers Fluorescent molecules, for
immunofluorescence
Enzymes for immunoblotting or ELISA
Fluorescence Recovery After Photobleach - FRAP - Answers Fluorescing label in cell
Shine UV light to spot to bleach
See how fast the label can recover, how the label moves into the bleached area
Resolution - Answers Also a key challenge in microscopy
,Physical limit of resolution for a light microscope is ~0.2 micrometers
Visible light is 0.38-0.75 micrometers
Super resolution - 30-50 n
EM - 0.1 nm
Transmission Electron Microscopy - TEM - Answers Roughly equivalent to light microscopy
Uses electrons instead of a light source
Need a column of vacuums so the electrons can move straight down
Advantages of EM - Answers EM has a resolution down to ~0.1 nm
-can see ribosomes, and large
protein complexes
-membrane bilayer is ~5 nm wide
Disadvantages of EM - Answers EM techniques require fixed samples
-use of electrons requires sample
observed to be in a vacuum
-optimal imaging requires use of heavy
metal stains to provide contrast
Scanning Electron Microscopy - Answers Collects reflected electrons off surface of samples
Spray heavy metal, electrons shoot on surface and reflect back
Cro-EM and immunogold labeling - Answers Used in electron microscopy
, Secondary antibody has gold particle
Gold particle is electron dense and can be seen as a black dot in the microscope
Different antibodies can have diff sized gold particles to recognize diff proteins
Monoclonal antibody production - Answers Inject mouse with antigen
Antigen causes immune response in mouse
Makes antibodies
Polyclonal antibodies - Answers Because the mouse can't be kept alive forever, can't keep
making antibody
Fuse with tumor cell that divides forever, makes hybridoma cells that divide and make the
antibody
Production of hybrid cells - Answers Centrifuge differentiated normal cell with tumor cell with a
fusing agent
Formation of heterokaryons, which are cultures
Selective medium allows only heterokaryons to survive and proliferate
Eventually get hybrid cells which are cloned
Could get different clones that recognize different parts of protein
Differential centrifugation - Answers Isolate different components of cells
Normal tissue culture cell size - Answers Normally 20-30 µm in diameter
Size of most eukaryotic cells - Answers 5 -100 µm
Kinds of light microscopy - Answers Transmission
Fluorescence
Transmission microscopy - Answers White light that has passed through the sample is
collected at the eyepiece
Epifluorescence microscopy - Answers Specimen is illuminated with the light of a specific
wavelength. This light is reabsorbed and only re-emitted fluorescence of a specific wavelength
is collected at the eyepiece.
Contrast - Answers Major challenge in transmission microscopic techniques
Because individual cells are predominantly transparent at high levels of magnification
Bright field - Answers Light transmitted straight through the specimen
Phase contrast - Answers Phase alterations of light transmitted through the specimen are
translated into brightness changes
Can see details
DIC - Answers Highlights edges where there is a steep change of refractive index
Different wavelengths
Dark field - Answers Specimen lit from side and only scattered light is seen
Used to look at fluorescence
Staining of cell components - Answers Because you can't see details by just microscopy alone
Paraffin section of urine-collecting ducts are stained with H&E
,Hematoxylin - Answers Basic dye that stains the nucleus
Eoisin - Answers Acidic dye that stains the cytosol
Fluorescence microscopy - Answers First barrier filers - only allows blue light through from light
source
Beam-splitting mirror - reflects light below 510 nm but transmits light above it
Second barrier filter - passes green light only to eyepiece
Multi-probe fluorescence microscopy - Answers Often antibodies are used to recognize a
protein, located on a particular structure, followed by a fluorescent second antibody
Can reveal subcellular compartments and cell dynamics
Primary antibody - Answers Rabbit antibody directed against antigen
Not usually labeled
Secondary antibody - Answers Marker-coupled antibody directed against rabbit antibodies
Kinds of tags that can be coupled to secondary antibodies - Answers Fluorescent molecules, for
immunofluorescence
Enzymes for immunoblotting or ELISA
Fluorescence Recovery After Photobleach - FRAP - Answers Fluorescing label in cell
Shine UV light to spot to bleach
See how fast the label can recover, how the label moves into the bleached area
Resolution - Answers Also a key challenge in microscopy
,Physical limit of resolution for a light microscope is ~0.2 micrometers
Visible light is 0.38-0.75 micrometers
Super resolution - 30-50 n
EM - 0.1 nm
Transmission Electron Microscopy - TEM - Answers Roughly equivalent to light microscopy
Uses electrons instead of a light source
Need a column of vacuums so the electrons can move straight down
Advantages of EM - Answers EM has a resolution down to ~0.1 nm
-can see ribosomes, and large
protein complexes
-membrane bilayer is ~5 nm wide
Disadvantages of EM - Answers EM techniques require fixed samples
-use of electrons requires sample
observed to be in a vacuum
-optimal imaging requires use of heavy
metal stains to provide contrast
Scanning Electron Microscopy - Answers Collects reflected electrons off surface of samples
Spray heavy metal, electrons shoot on surface and reflect back
Cro-EM and immunogold labeling - Answers Used in electron microscopy
, Secondary antibody has gold particle
Gold particle is electron dense and can be seen as a black dot in the microscope
Different antibodies can have diff sized gold particles to recognize diff proteins
Monoclonal antibody production - Answers Inject mouse with antigen
Antigen causes immune response in mouse
Makes antibodies
Polyclonal antibodies - Answers Because the mouse can't be kept alive forever, can't keep
making antibody
Fuse with tumor cell that divides forever, makes hybridoma cells that divide and make the
antibody
Production of hybrid cells - Answers Centrifuge differentiated normal cell with tumor cell with a
fusing agent
Formation of heterokaryons, which are cultures
Selective medium allows only heterokaryons to survive and proliferate
Eventually get hybrid cells which are cloned
Could get different clones that recognize different parts of protein
Differential centrifugation - Answers Isolate different components of cells
