Microscopy and Lab techniques

Question 1

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

Answer 1

1) fix
2) stain

Question 2

Fixation

Answer 2

• 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.

Question 3

Staining

Answer 3

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

Question 4

General Types of Microscopy

Answer 4

1) Optical microscopy
2) electron microscopy

Question 5

Optical microscopy

Answer 5

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

Question 6

Types of Optical Microsope

Answer 6

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

Question 7

Stereo microscopes (dissection microscopes):

Answer 7

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

Question 8

Compound microscopes

Answer 8

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

Question 9

Bright field microscopes

Answer 9

• compound
  microscopes with a bright light.

Question 10

Phase contrast microscopes

Answer 10

• 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).

Question 11

Fluorescence microscopy

Answer 11

• 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.

Question 12

Confocal laser scanning microscopy

Answer 12

• 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.

Question 13

Dark field microscopy

Answer 13

• 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.

Question 14

Types of Electron Microscope

Answer 14

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

Question 15

Scanning electron microscopy (SEM)

Answer 15

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

Question 16

Cryo-scanning electron microscopy
(cryo-SEM)

Answer 16

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

Question 17

Transmission electron microscopy (TEM)

Answer 17

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

Question 18

Electron tomography

Answer 18

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

Question 19

Techniques to Count Cells

Answer 19

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

Question 20

Hemocytometers (counting chambers):

Answer 20

-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.

Question 21

Colony Forming Units (CFUs)

Answer 21

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

Question 22

Automated cell counting

Answer 22

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

Question 23

Phases of Bacterial Growth Curve

Answer 23

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

Question 24

Cell fractionation

Answer 24

• 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.

Question 25

Differential centrifugation

Answer 25

- 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.

Question 26

Density centrifugation

Answer 26

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

Question 27

Blood centrifugation

Answer 27

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

Question 28

Lab Techs for Nucleic Acid and Proteins

Answer 28

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

Question 29

Karyotyping

Answer 29

- 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).

Question 30

DNA sequencing:

Answer 30

- 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.

Question 31

Dideoxynucleotides (ddNTPs)

Answer 31

- 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.

Question 32

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

Answer 32

1) single nucleotide polymorphism (SNPs) ***Refer to page 52 DAT Bootcamp for image***

Question 33

Recombinant DNA

Answer 33

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

Question 34

Restriction fragment length polymorphisms (RFLPs)

Answer 34

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

Question 35

DNA fingerprinting:

Answer 35

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

Question 36

CRISPR:

Answer 36

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

Question 37

Polymerase Chain Reaction (PCR):

Answer 37

- automated process creating millions of copies of DNA

Question 38

3 Steps in PCR:

Answer 38

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.

Question 39

Bacterial cloning:

Answer 39

- 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.

Question 40

Gel electrophoresis:

Answer 40

- 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.

Question 41

Sodium dodecyl sulfate (SDS)

Answer 41

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

Question 42

Southern blotting:

Answer 42

- 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.

Question 43

Northern blotting:

Answer 43

- identifying fragments of known RNA using an RNA probe.

Question 44

Western blotting:

Answer 44

- 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

Question 45

Enzyme-Linked Immunosorbent Assay (ELISA):

Answer 45

- 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.

Question 46

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.

Answer 46

- 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

Question 47

Gene therapy:

Answer 47

- 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.

Question 48

Genomics

Answer 48

- is the study of all genes present in an organism’s genome and how they interact.

Question 49

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.

Answer 49

1) Genome annotation

Question 50

A genomic library

Answer 50

- 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.

Question 51

DNA microarrays

Answer 51

- 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.

Question 52

Transgenic animals

Answer 52

- 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.

Question 53

Reproductive cloning:

Answer 53

- 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

Question 54

Totipotent cells:

Answer 54

- can differentiate into an entire organism (including extraembryonic membranes). E.g. zygote → morula.

Question 55

Pluripotent cells:

Answer 55

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

Question 56

Multipotent cells:

Answer 56

- can give rise to some of the three germ layers - not all.

Question 57

Chromatography:

Answer 57

- 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.

Question 58

Fluorescence Recovery After Photobleaching (FRAP):

Answer 58

- 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.

Question 59

Fluorescence Lifetime Imaging Microscopy (FLIM):

Answer 59

- 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.

Question 60

Immunofluorescence microscopy:

Answer 60

- A technique that identifies the localization of proteins of interest, using fluorophores.

Question 61

Knockout mice:

Answer 61

- selected gene is ‘knocked out’ and changes between knockout and wild type are observed.

Question 62

Serial Dilution:

Answer 62

- 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/[ ]