DAT bio Chapter 8 Microscopes

Question 1

What is fixation

Answer 1

getting cells to stick to the slide and preserving them in their most life like state

Question 2

2 types of fixation

Answer 2

heat and chemical

Question 3

heat fixation

Answer 3

put cell on slide
run the bottom of the slide over a bunsen burner.
This heats the cell, preserving and sticking them to the slide

Question 4

What does staining do

Answer 4

adds color to cells helping us see their structures easier (down side) it kills the cell

Question 5

Optical microscopy

Answer 5

Cells viewed directly
light shines on sample
used to observe living cells

Question 6

Electron microscopy:

Answer 6

Cells viewed indirectly using a computer after it is bombarded with electrons that passes through a magnetic field in a vacuum. Used to see smaller objects.

Question 7

Requirement to see cells on electron microscope

Answer 7

cells must be fixed, stained, and killed

Question 8

Stereo microscopes (

Answer 8

(dissection microscopes):

use low magnification to view the surface of an object

Question 9

Compound microscopes

Answer 9

multiple lenses to view sample. cells are one cell thick, and alive.
Cons
without fixing and staining, they have poor contrast. which sucks because fixing and staining would mean the cells would die

Question 10

example of compound microscope

Answer 10

bright field microscopes

Question 11

Bright field microscopes:

Answer 11

compound microscopes with a bright light

Question 12

Phase contrast microscopes:

Answer 12

view: thin samples with live cells.

Due to crazy good contrast, cells are not fixed or stained

Question 13

How does phase contrast microscopes achieve its high contrast?

Answer 13

Light is refracted
through an annular ring creating a phase shift,
leading to high contrast. Large phase shifts can
lead to a halo effect.

Question 14

How can you reduce halo effects in phase contrast microscopes?

Answer 14

can be reduced with

phase plates or thinner samples

Question 15

Fluorescence microscopy

Answer 15

: fluorophores
( fluorescent chemical that will re-emit light upon being excited by another light source) are attached to parts of a specimen. Using different types of fluorophores allows researchers to view 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 in the image (artifacts) decrease the resolution.

Question 16

Confocal laser scanning microscopy:

Answer 16

visualizes fluorescent objects in living cells. Can be used
without fluorescence tagging. Artifacts (distortion in the image) are
reduced by focusing a beam of UV light onto the
sample. This reduces intensity so samples must
be illuminated longer

Question 17

Confocal laser scanning microscopy was made to overcome _____

Answer 17

artifacts or distortions of fluorescence microscopy.

Question 18

Dark field microscopy

Answer 18

: 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 19

Scanning electron microscopy (SEM)

Answer 19

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

Question 20

Cryo-scanning electron microscopy

Answer 20

type of SEM where the sample is
frozen in liquid nitrogen instead of dehydrated.
Costly and produces artifacts (distortions in the image).

Question 21

Transmission electron microscopy (TEM)

Answer 21

high resolution 2D images of the sample’s

internal structures.

Question 22

Electron tomography

Answer 22

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

Question 23

3 techniques to count cells

Answer 23

Hemocytometers (counting chambers):
Colony Forming Units (CFUs):
Automated cell counting

Question 24

Hemocytometers

Answer 24

(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

Question 25

Colony Forming Units (CFUs):

Answer 25

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

Question 26

Automated cell counting

Answer 26

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 27

Electrical resistance does what

Answer 27

stops electrical conduction, when this happens, we can count the cells that are stopping the electrical conduction

Question 28

Bacterial growth curve

Answer 28

describe the growth pattern of a given culture of cells

Question 29

Bacterial growth curve has 4 stages

Answer 29

1. Lag phase: Adaptation prior to cell division 2. Exponential (log) phase: Rapid doubling 3. Stationary phase: Growth rate = death rate 4. Death phase: Decline due to lack of food/other variable

Question 30

Cell fractionation

Answer 30

separates cell | contents by centrifugation

Question 31

what is centrifuge

Answer 31

``` 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 32

Differential centrifugation

Answer 32

cells must first be split open so that the components can be separated. (homogenization). Multiple cycles where supernatant is removed and spun again allow for fractionation (isolation) of each organelle.

Question 33

Process called where cells must first be split open so that the components can be separated

Answer 33

homogenization

Question 34

What is supernatant

Answer 34

clear liquid that lies above the solid residue after centrifugation,

Question 35

Density centrifugation:

Answer 35

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

Question 36

Karyotyping:

Answer 36

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 37

DNA sequencing

Answer 37

sequencing nucleotides from cut parts (fragments) of the DNA.. Can sequence complete genomes piece by piece. In humans single nucleotide polymorphisms (SNPs) serve as markers for disease causing genes

Question 38

2 methods for DNA sequencing

Answer 38

1) dideoxy chain termination (Sanger sequencing) older and more established method) 2) next generation sequencing. (newer)

Question 39

When is recombinant DNA produced?

Answer 39

when DNA fragments from different sources are joined together. These fragments are produced by restriction enzymes, which tend to cut DNA at palindromic (sequence that reads the same backward as forward... AAT TAA), sequences to produce sticky (unpaired nucleotides) or blunt ends (paired nucleotides).

Question 40

Restriction fragment length | polymorphisms (RFLPs) function

Answer 40

unique lengths of DNA from restriction enzymes; allow for comparison between individuals by analyzing non-coding DNA (coding DNA is highly conserved). Not different? so wont do us any good in identifying individuals?

Question 41

DNA fingerprinting

Answer 41

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

Question 42

CRISPR

Answer 42

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

Question 43

Polymerase Chain Reaction (PCR):

Answer 43

automated | process creating millions of copies of DNA

Question 44

3 steps for PCR

Answer 44

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 45

Bacterial cloning

Answer 45

cloning eukaryotic gene products in prokaryotic cells. Used to produce medicine

Question 46

Steps (protocol for bacterial cloning) PART 1

Answer 46

Processed mRNA for eukaryotic gene is isolated then treated with reverse transcriptase to make cDNA

Question 47

Steps (protocol for bacterial cloning) PART 2

Answer 47

``` 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) ```

Question 48

Steps (protocol for bacterial cloning) PART 3

Answer 48

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 49

Gel electrophoresis

Answer 49

: 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 50

Southern blotting:

Answer 50

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 51

Northern blotting

Answer 51

identifying fragments of | known RNA using an RNA probe.

Question 52

Western blotting:

Answer 52

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).

Question 53

What is a DNA probe?

Answer 53

NA probes are single stranded DNA, so they only hybridize with complementary DNA sequences.

Question 54

1. Enzyme-Linked Immunosorbent Assay | (ELISA):

Answer 54

: 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 55

Pulse chase experiments:

Answer 55

useful for researchers that want to know more about how proteins move through a cell. This is beneficial because it gives researchers information about gene expression for any given cell type. Also, it illustrates the fate of those same gene products (proteins). 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

Question 56

Genomics

Answer 56

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

Question 57

genomic library

Answer 57

stores the DNA of an | organism’s genome

Question 58

DNA microarrays

Answer 58

contain thousands of DNA probes that bind to complementary DNA fragments, allowing researchers to see which genes are expressed.

Question 59

Protocol for DNA microarrays

Answer 59

``` isolate a cell and remove mRNA (because it represents the active transcription of that cell type)→ synthesize cDNA from mRNA using reverse transcriptase → hybridize cDNA with DNA probes → examine microarray for fluorescence → compare microarray with the sequenced genome ```

Question 60

Transgenic animals

Answer 60

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 61

Reproductive cloning:

Answer 61

producing a genetic copy of an organism from a somatic cell (any cell of a living organism other than the reproductive cells) . A multipotent cell must be converted to a totipotent cell. E.g. Dolly the sheep

Question 62

Reproductive cloning | - totipotent cells

Answer 62

Single cell with the ability to divide and produce an entire organism. . E.g. zygote → morula.

Question 63

Pluripotent cells:

Answer 63

Stem cell that can differentiate into any of the three germ layers: endoderm, mesoderm, or ectoderm. Cannot develop an entire organism because they cant develop extraembryonic tissue, like the placenta.

Question 64

Multipotent cells

Answer 64

can give rise to some of | the three germ layers - not all. These cells are most differentiated. cannot develop entire organisms

Question 65

Chromatography:

Answer 65

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 66

Fluorescence Recovery After | Photobleaching (FRAP):

Answer 66

: quantitative measure of how and where biomolecules move in a live cell.

Question 67

Fluorescence Recovery After | Photobleaching (FRAP): protocol

Answer 67

``` baseline fluorescence is measured → area of the sample is photobleached (Photobleaching causes pigmented molecules to irreversibly lose their fluorescence.) → photobleached molecules are replaced by unbleached molecules overtime due to cell dynamics → area gradually recovers fluorescence. ```

Question 68

2 types of live cell visualization that utilize fluorescence.

Answer 68

FRAP and FLIM

Question 69

Fluorescence Lifetime Imaging Microscopy | (FLIM):

Answer 69

provides a quantitative measure of the concentration of various ions, molecules, and gases in a cell. Cell is irradiated with light and fluorescence lifetime (amount of time it takes for an exited molecule to release all its fluorescence) is measured

Question 70

Knockout mice:

Answer 70

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