Microscopy and Lab techniques Flashcards
Before we can use microscopy, we must first ____ and _____ cells:
1) fix
2) stain
Fixation
- 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
the slide.
Staining
- adds color to cells, making cell structures easier to visualize. Staining often kills
the cells.
General Types of Microscopy
1) Optical microscopy
2) electron microscopy
Optical microscopy
- cells are viewed directly.
Light shines on a sample and is magnified via
lenses. Can be used to observe living cells.
Types of Optical Microsope
1) Stereo microscopes
2) Compound microscopes
3) Bright field microscopes
4) Phase contrast microscopes
5) Fluorescence microscopy
6) Confocal laser scanning microscopy
7) Dark field microscopy
Stereo microscopes (dissection microscopes):
- Use low magnification to view the surface of an
object.
Compound microscopes
- have multiple lenses
to view simple, one-cell thick, live cells. Without
fixing and staining, they have poor contrast.
Bright field microscopes
- compound
microscopes with a bright light.
Phase contrast microscopes
- 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).
Fluorescence microscopy
- fluorophores
(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
- increases contrast
between the 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.
Types of Electron Microscope
1) Scanning electron microscopy (SEM)
2) Cryo-scanning electron microscopy
(cryo-SEM)
3) Transmission electron microscopy (TEM)
4) Electron tomography
Scanning electron microscopy (SEM)
- high resolution 3D images of the surface of a
dehydrated sample.
Cryo-scanning electron microscopy
(cryo-SEM)
- type of SEM where the 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
internal structures.
Electron tomography
- not a type of
microscopy. Sandwiches TEM images to create
a 3D image of the sample’s internal structure.
Techniques to Count Cells
1) Hemocytometers
2) Colony Forming Units (CFUs)
3) Automated cell counting
Hemocytometers (counting chambers):
-gridded slide under microscope. Cells can be
counted in a known area, and that number can
be extrapolated to find the full volume of the
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 pass through a narrow tube and are detected by
laser).
Phases of Bacterial Growth Curve
1) Lag phase - Adaptation prior to cell division
2) Exponential Phase -Rapid doubling
3) Stationary Phase -Growth rate = death rate
4) Death Phase - Decline due to lack of food/other variable
Cell fractionation
- separates cell contents by
centrifugation. A centrifuge spins contents to
separate them by mass, density, and/or shape. More
dense particles collect at the bottom (pellet) and
less dense particles remain as supernatant liquid
on top.
Differential centrifugation
- cells are first split
open to release contents (homogenization).
Multiple cycles where supernatant is removed
and spun again allow for fractionation
(isolation) of each organelle.
Density centrifugation
- one cycle where
organelles are separated by density into layers. - From most dense to least dense: nuclei >
mitochondria/chloroplast > ER fragments >
ribosomes
Blood centrifugation
- is a type of density
centrifugation with 3 layers: plasma (clear
fluid), buffy coat ( thin layer composed of
leukocytes and platelets), and erythrocytes.
Lab Techs for Nucleic Acid and Proteins
1) Karyotyping
2) DNA Sequencing
3) DNA fingerprinting
4) CRISPR
5) Polymerase Chain Reaction (PCR)
6) Bacterial Cloning
7) Gel Electrophoresis
8) Southern Blotting
9) Northern Blotting
10) Western Blotting
11) Enzyme-linked Immunosorbent Assay (ELISA)
12) Pulse Chase Experiments
13) Gene Therapy
Karyotyping
- observing chromosomes under light microscope during metaphase. Can be
used to diagnose conditions involving chromosomal aberrations, breakages, or
aneuploidies (e.g. Down’s syndrome or trisomy
21).
DNA sequencing:
- sequencing nucleotides in
fragments of DNA. 2 methods are dideoxy chain termination (Sanger sequencing)
(older) and next generation sequencing (newer). Can sequence complete genomes
piece by piece. Both methods may use shotgun sequencing, where cloned DNA genomes are cut into pieces that are sequenced and recompiled to observe
sequence overlaps.
Dideoxynucleotides (ddNTPs)
- used in Sanger
sequencing lack two hydroxyl (OH-) groups. These
ddNTPs are mixed with normal dNTPs nucleotides
for DNA Pol to use. When added to a DNA strand,
ddNTPs result in the termination of elongation
because they lack a 3’ OH- needed for new
phosphodiester bond formation with other
nucleotides.
In humans, _______ serve as markers for disease causing genes.
1) single nucleotide polymorphism (SNPs)
Refer to page 52 DAT Bootcamp for image
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
polymorphisms (RFLPs)
- are unique
lengths of DNA from restriction enzymes;
they allow for comparison between individuals by analyzing non-coding DNA
(coding DNA is highly conserved).
DNA fingerprinting:
- identifies individuals
through unique aspects of DNA such as RFLPs
and short tandem repeats (STR’s). Used in
paternity and forensic cases.
CRISPR:
- used to edit specific genomic regions
of interest by adding or deleting specific
targeted sequences of DNA. Used in gene
therapy.
Polymerase Chain Reaction (PCR):
- automated process creating millions of copies of DNA
3 Steps in PCR:
I. 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 polymerase.
Bacterial cloning:
- cloning eukaryotic gene
products in prokaryotic cells. Used to produce
medicine. - Protocol: Processed mRNA for eukaryotic gene is isolated then treated with reverse transcriptase to make cDNA → cDNA incorporated into plasmid (transfer vector) using restriction enzymes 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.
Gel electrophoresis:
- 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.
Sodium dodecyl sulfate (SDS)
- A strong
detergent used in gel electrophoresis to denature,
linearize, and add a negative charge to proteins to
separate them by size and charge.
Southern blotting:
- identifies fragments of
known DNA sequence in a large population of
DNA. Electrophoresed DNA is separated into
single strands and identified via complementary
DNA probes.
Northern blotting:
- identifying fragments of
known RNA using an RNA probe.
Western blotting:
- 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
antibody).
SNOW DROP
Southern - DNA
Northern - RNA
nothing - nothing
Western - protein
Enzyme-Linked Immunosorbent Assay
(ELISA):
- determines if a person has a specific antigen. Important to diagnose diseases (e.g.
HIV). Antibodies are placed on a microtiter
plate with a sample and change color if
antigens are present.
Pulse chase experiments:
During the _____ amino acids are radioactively labeled and then incorporated
into proteins. The ______ prevents radioactively labeled protein production. Using simple staining, the radioactive proteins can be
tracked.
- useful for studying
gene expression and the fate of proteins by
viewing how a protein moves through a cell.
1) Pulse Phase
2) Chase phase
Gene therapy:
- The process of inserting genes into a cell using viral or non-viral methods.
Viruses are the preferred method to insert
genes into a cell because they have the highest
transduction efficiency. However, viruses
may cause an immune response. Non-viral
methods do not cause an immune response,
but are less efficient at inserting genes.
Genomics
- is the study of all genes present in an
organism’s genome and how they interact.
After a gene has been sequenced, it must be
annotated via ________. This process
identifies the location of genes and coding regions
in a genome, and determines each of their
functions.
1) Genome annotation
A genomic library
- 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 can then be
cloned via bacterial cloning.
DNA microarrays
- 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
genome.
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.
Reproductive cloning:
- 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.
1) Totipotent
2) Pluripotent
3) Multipotent
Totipotent cells:
- can differentiate into an
entire organism (including extraembryonic
membranes). E.g. zygote → morula.
Pluripotent cells:
- can differentiate into
the three germ layers (endoderm,
mesoderm, ectoderm). Cannot give rise to
extraembryonic membranes.
Multipotent cells:
- can give rise to some of
the three germ layers - not all.
Chromatography:
- 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
heights.
Fluorescence Recovery After
Photobleaching (FRAP):
- quantitative measure
of how and where biomolecules move in a live
cell.
● 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
(FLIM):
- provides a quantitative measure of the
concentration of various ions, molecules, and gases
in a cell. Cells are irradiated with light and
fluorescence lifetime is measured.
Immunofluorescence microscopy:
- A
technique that identifies the localization of
proteins of interest, using fluorophores.
Knockout mice:
- selected gene is ‘knocked out’ and changes between knockout and wild type are
observed.
Serial Dilution:
- decreasing the concentration
of solute in a solution by a dilution factor. Used
to decrease bacterial concentrations to a
testable concentration.
[ ] = (1/n)^#steps
n = dilution ratio
# steps = number of times the serial dilution was repeated
Dilution = 1/[ ]
