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Microscopy and Lab Techniques
Terms in this set (45)
Getting cells to 'stick' to the slide and
preserving them in their most life-like state.
There are 2 types: heat fixation and chemical
fixation. During heat fixation, cells are placed
on top of the slide and then the underside of
the slide is run over a bunsen burner. This
heats the cells, preserving and sticking them to
Staining adds color to cells, making cell
structures easier to visualize. Staining often kills
Cells are viewed directly.
Light shines on a sample and is magnified via
lenses. Can observe living cells.
Cells are viewed indirectly
via computer after being
bombarded with electrons
which pass through
magnetic fields in a
vacuum. Can be used to
view smaller objects but
cells must be fixed,
stained (metal coated) and
Stereo microscopes (dissection microscopes)
Low magnification to view surface of an object.
Have multiple lenses
to view simple, one-cell thick, live cells. Without
fixing and staining, it has poor contrast.
Bright Field Microscopes
Compound microscopes with a bright light.
Phase Contrast Microscope
Can view thin
samples with live cells. Light is refracted
through an annular ring creating a phase shift,
leading to high contrast. Large phase shifts can
lead to a halo effect (can be reduced with
phase plates or thinner samples).
(fluorescent chemicals) are used to visualize
different parts of the cell. A dichroic filter is
used which allows certain wavelengths of light
to be reflected and others to pass through.
Distortions or artifacts decrease the resolution.
Confocal Laser Scanning Microscopy
Visualizes fluorescent objects. Can be used
without fluorescence tagging. Artifacts are
reduced by focusing a beam of UV light onto the
sample. This reduces intensity so samples must
be illuminated longer.
Dark Field Microscopy
between sample and the field around it to
allow visualization of unstained live cells. Only
scattered light is viewed - allows the sample to
be viewed against a black background.
Scanning Electron Microscopy (SEM)
High resolution 3D images of the surface of a
Cryo-scanning Electron Microscopy
Type of SEM where sample is
frozen in liquid nitrogen instead of dehydrated.
Costly and produces artifacts.
Transmission Electron Microscopy (TEM):
High resolution 2D images of the sample's
Not a type of
microscopy. Sandwiches TEM images to create
a 3D image of sample's internal structure.
Hemocytometers (counting chambers)
Gridded slide under microscope. Can count
cells in a known area and extrapolate for full
volume of sample.
Colony Forming Units (CFUs)
Estimates number of cells plated on growth medium assuming that one cell gives rise to one colony.
Automated Cell Counting
Includes electrical resistance (counting cells by observing flow of electricity) and flow cytometry (cells in narrow tube detected by laser).
Separates cell contents by
Cells are first split open to release contents (homogenization). Multiple cycles where supernatant is removed and spun again allowing for fractionation (isolation) of each organelle.
One cycle where
organelles are separated by density into layers. From most dense to least dense: nuclei >
mitochondria/chloroplast > ER fragments >
Observing chromosomes under
light microscope during metaphase. Can be
used to diagnose conditions involving
chromosomal aberrations, breakages,
aneuploidies (e.g. Down's syndrome or trisomy
Sequencing nucleotides in
fragments of DNA. 2 methods are dideoxy
chain termination or Sanger sequencing
(older) and next generation sequencing
(newer). Can sequence complete genomes
piece by piece. In humans single nucleotide
polymorphisms (SNPs) serve as markers for
disease causing genes.
Recombinant DNA is produced when
restriction enzymes cut DNA at
palindromic sequences generating sticky
ends (have unpaired nucleotides) or blunt
ends (have paired nucleotides).
Restriction Fragment Length
Unique lengths of DNA from restriction enzymes,
allows for comparison between individuals.
Analyzes non-coding DNA (coding DNA is
Identifies individuals through unique aspects of DNA such as RFLPs and short tandem repeats (STR's). Used in paternity and forensic cases.
Polymerase Chain Reaction (PCR)
1. Denaturation (~95 °C): heating separates
DNA into single strands.
II. Primer annealing (~65 °C): DNA primers
hybridize with single strands.
III. Elongation (~70 °C): nucleotides are added
to the 3' end of DNA using Taq
Cloning eukaryotic gene
products in prokaryotic cells. Used to produce
Protocol: Processed mRNA for eukaryotic
gene is isolated then treated with reverse
transcriptase to make cDNA → cDNA
incorporated into plasmid (transfer
vector) using reverse transcriptase and
DNA ligase → vector taken up by
competent bacterial cells (can undergo
transformation; made competent using
electroporation or heat shock) and undergo
transformation → gene of interest is
found using antibiotic resistance
(antibiotic resistant gene attached to target
gene) or color change (vectors containing
genes making cells blue) methods.
Separates DNA fragments by charge and size. An electric field is applied to agarose gel (top = negative cathode, bottom = positive anode). Smaller fragments travel further from top of gel.
Identifies fragments of known DNA sequence in a large population of DNA. Electrophoresed DNA separated into single strands and identified via complementary DNA probe.
Identifies fragments of
known RNA using an RNA probe.
Quantifies amount of target
protein in a sample using sodium dodecyl
sulfate polyacrylamide gel electrophoresis or
SDS PAGE (proteins denatured and given
negative charge proportional to their mass).
Treated with primary antibody (binds to
target protein) and secondary antibody
(attached to indicator and binds to primary
Enzyme-Linked Immunosorbent Assay
Determines if a person has a specific
antigen. Important to diagnose diseases (e.g.
HIV). Antibodies are placed on a microtiter
plate and with antigens and change color.
Pulse Chase Experiments
Useful for studying
gene expression and the fate of proteins by
viewing how a protein moves through a cell.
During the pulse phase amino acids are
radioactively labeled and then incorporated
into proteins. The chase phase prevents
radioactively labelled protein production.
Using simple staining, the radioactive proteins
can be tracked.
Stores the DNA of an
organism's genome. DNA fragments are
incorporated into plasmids and can be
screened for by using antibiotic resistance and
color changing techniques. They are then
cloned via bacterial cloning.
Contain thousands of DNA
probes that bind to complementary DNA
fragments, allowing researchers to see which
genes are expressed.
Protocol: isolate a cell and remove mRNA
(active transcription) → synthesize cDNA
from mRNA using reverse transcriptase →
hybridize cDNA with DNA probes →
examine microarray for fluorescence →
compare microarray with the sequenced
Transgenic animals are models used to
identify the function of a gene. A gene is taken
from one organism and inserted into another.
Can be used for mass medication production
(e.g. clotting factors for hemophiliacs). This
process is labor intensive.
Producing a genetic
copy of an organism from a somatic cell. A
multipotent cell must be converted to a
totipotent cell. E.g. Dolly the sheep.
Can differentiate into an
entire organism (including extraembryonic
membranes). E.g. zygote → morula.
Can differentiate into
the three germ layers (endoderm,
mesoderm, ectoderm). Cannot give rise to
Can give rise to some of
the three germ layers - not all.
Separating components of
a heterogeneous sample using differential
solubility. The sample is dissolved in the solvent
(mobile phase) and placed in an apparatus
containing the stationary phase. The mobile
phase climbs up the stationary phase and the
different components ascend to different
Fluorescence Recovery After
of how and where biomolecules move in a live
Protocol: baseline fluorescence is
measured → area of the sample is
photobleached → photobleached molecules
are replaced by unbleached molecules
overtime due to cell dynamics → area
gradually recovers fluorescence.
Fluorescence Lifetime Imaging Microscopy
Provides a quantitative measure of the
concentration of various ions, molecules, and gases in a cell. Cell is irradiated with light and fluorescence lifetime is measured.
Selected gene is 'knocked out'
and changes between knockout and wild type
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