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Microscopy and Lab techniques Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

Before we can use microscopy, we must first ____ and _____ cells:

A

1) fix
2) stain

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2
Q

Fixation

A
  • 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.
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3
Q

Staining

A
  • adds color to cells, making cell structures easier to visualize. Staining often kills
    the cells.
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4
Q

General Types of Microscopy

A

1) Optical microscopy
2) electron microscopy

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5
Q

Optical microscopy

A
  • cells are viewed directly.
    Light shines on a sample and is magnified via
    lenses. Can be used to observe living cells.
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6
Q

Types of Optical Microsope

A

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

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7
Q

Stereo microscopes (dissection microscopes):

A
  • Use low magnification to view the surface of an
    object.
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8
Q

Compound microscopes

A
  • have multiple lenses
    to view simple, one-cell thick, live cells. Without
    fixing and staining, they have poor contrast.
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9
Q

Bright field microscopes

A
  • compound
    microscopes with a bright light.
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10
Q

Phase contrast microscopes

A
  • 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).
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11
Q

Fluorescence microscopy

A
  • 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.
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12
Q

Confocal laser scanning microscopy

A
  • 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.
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13
Q

Dark field microscopy

A
  • 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.
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14
Q

Types of Electron Microscope

A

1) Scanning electron microscopy (SEM)
2) Cryo-scanning electron microscopy
(cryo-SEM)
3) Transmission electron microscopy (TEM)
4) Electron tomography

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15
Q

Scanning electron microscopy (SEM)

A
  • high resolution 3D images of the surface of a
    dehydrated sample.
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16
Q

Cryo-scanning electron microscopy
(cryo-SEM)

A
  • type of SEM where the sample is frozen in liquid nitrogen instead of dehydrated. Costly and produces artifacts.
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17
Q

Transmission electron microscopy (TEM)

A
  • high resolution 2D images of the sample’s
    internal structures.
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18
Q

Electron tomography

A
  • not a type of
    microscopy. Sandwiches TEM images to create
    a 3D image of the sample’s internal structure.
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19
Q

Techniques to Count Cells

A

1) Hemocytometers
2) Colony Forming Units (CFUs)
3) Automated cell counting

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20
Q

Hemocytometers (counting chambers):

A

-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.

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21
Q

Colony Forming Units (CFUs)

A
  • estimates
    number of cells plated on growth medium
    assuming that one cell gives rise to one colony.
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22
Q

Automated cell counting

A
  • includes electrical
    resistance (counting cells by observing flow of electricity) and flow cytometry (cells pass through a narrow tube and are detected by
    laser).
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23
Q

Phases of Bacterial Growth Curve

A

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

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24
Q

Cell fractionation

A
  • 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.
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25
Q

Differential centrifugation

A
  • 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.
26
Q

Density centrifugation

A
  • one cycle where
    organelles are separated by density into layers.
  • From most dense to least dense: nuclei >
    mitochondria/chloroplast > ER fragments >
    ribosomes
27
Q

Blood centrifugation

A
  • is a type of density
    centrifugation with 3 layers: plasma (clear
    fluid), buffy coat ( thin layer composed of
    leukocytes and platelets), and erythrocytes.
28
Q

Lab Techs for Nucleic Acid and Proteins

A

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

29
Q

Karyotyping

A
  • 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).
30
Q

DNA sequencing:

A
  • 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.
31
Q

Dideoxynucleotides (ddNTPs)

A
  • 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.
32
Q

In humans, _______ serve as markers for disease causing genes.

A

1) single nucleotide polymorphism (SNPs)

Refer to page 52 DAT Bootcamp for image

33
Q

Recombinant DNA

A
  • is produced when
    restriction enzymes cut DNA at palindromic sequences, generating sticky
    ends (have unpaired nucleotides) or blunt
    ends (have paired nucleotides).
34
Q

Restriction fragment length
polymorphisms (RFLPs)

A
  • are unique
    lengths of DNA from restriction enzymes;
    they allow for comparison between individuals by analyzing non-coding DNA
    (coding DNA is highly conserved).
35
Q

DNA fingerprinting:

A
  • identifies individuals
    through unique aspects of DNA such as RFLPs
    and short tandem repeats (STR’s). Used in
    paternity and forensic cases.
36
Q

CRISPR:

A
  • used to edit specific genomic regions
    of interest by adding or deleting specific
    targeted sequences of DNA. Used in gene
    therapy.
37
Q

Polymerase Chain Reaction (PCR):

A
  • automated process creating millions of copies of DNA
38
Q

3 Steps in PCR:

A

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.

39
Q

Bacterial cloning:

A
  • 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.
40
Q

Gel electrophoresis:

A
  • 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.
41
Q

Sodium dodecyl sulfate (SDS)

A
  • A strong
    detergent used in gel electrophoresis to denature,
    linearize, and add a negative charge to proteins to
    separate them by size and charge.
42
Q

Southern blotting:

A
  • 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.
43
Q

Northern blotting:

A
  • identifying fragments of
    known RNA using an RNA probe.
44
Q

Western blotting:

A
  • 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

45
Q

Enzyme-Linked Immunosorbent Assay
(ELISA):

A
  • 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.
46
Q

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.

A
  • 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

47
Q

Gene therapy:

A
  • 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.
48
Q

Genomics

A
  • is the study of all genes present in an
    organism’s genome and how they interact.
49
Q

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.

A

1) Genome annotation

50
Q

A genomic library

A
  • 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.
51
Q

DNA microarrays

A
  • 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.

52
Q

Transgenic animals

A
  • 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.
53
Q

Reproductive cloning:

A
  • 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

54
Q

Totipotent cells:

A
  • can differentiate into an
    entire organism (including extraembryonic
    membranes). E.g. zygote → morula.
55
Q

Pluripotent cells:

A
  • can differentiate into
    the three germ layers (endoderm,
    mesoderm, ectoderm). Cannot give rise to
    extraembryonic membranes.
56
Q

Multipotent cells:

A
  • can give rise to some of
    the three germ layers - not all.
57
Q

Chromatography:

A
  • 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.
58
Q

Fluorescence Recovery After
Photobleaching (FRAP):

A
  • 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.
59
Q

Fluorescence Lifetime Imaging Microscopy
(FLIM):

A
  • 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.
60
Q

Immunofluorescence microscopy:

A
  • A
    technique that identifies the localization of
    proteins of interest, using fluorophores.
61
Q

Knockout mice:

A
  • selected gene is ‘knocked out’ and changes between knockout and wild type are
    observed.
62
Q

Serial Dilution:

A
  • 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/[ ]

Biology (15 decks)

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