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DAT BIOLOGY > ch 8- microscopy and lab techniques > Flashcards

ch 8- microscopy and lab techniques Flashcards

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1
Q

fixation

A

sticking cells to a slide

preserving them in their most lifelike state

chemical and heat fixation

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2
Q

what does staining do

A

often kills cells

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3
Q

electron microscopy

A

indirectly viewing cells through computer after bombarding them with electrons that pass through a magnetic field in a vaccuum

cells MUST be fixed and stained w metal coat and killed

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4
Q

stereo microscope

A

a dissection microscope

view surface of object

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5
Q

what can you see with a compound microscope

A

one cell thick live cells

have poor contrast without fixing and staining

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6
Q

phase contrast microscopy

A

viewing think samples with live cells

light refracted with annular ring creating phase shift
-can lead to halo effect

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7
Q

how to remove halo effect

A

phase plate or thinner samples

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8
Q

what is used in flourescence microscopy

A

dichroic filter
- allows certain wavelengths of light to be reflected and others to pass through

distortions or artifacts decrease resolution

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9
Q

confocal microscopy

A

visualizes flourescent objects

can be used without flouresence tagging

artifacts reduced by focusing beam of UV light on sample
-reduces intensity so samples must be illuminated longer

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10
Q

dark field micrsocopy

A

viewing unstained live cells bc the contrast is increased

only scattered light is viewed

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11
Q

scannning electron microscopy

A

high resolution 3D image of surface of DEHYDRATED sample

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12
Q

cryo SEM

A

sample frozen in liquid nitrogen instead of dehydrated

costly
produces artifacts

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13
Q

TEM

A

high resolution
2D images

internal structures

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14
Q

electron tomography

A

sandwiches TEM images to create 3D images of samples internal strutures

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15
Q

hemcytometer

A

counting chambers used to count cells

Gridded slide under microscope
-count cells in known area and extrapolate data

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16
Q

colony forming units (CFU)

A

estimate number of cells assuming one cell gives rise to one colony

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17
Q

automated cell counting

A

electrical resistance - counting cells by observing flow of elctricityf

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18
Q

flow cytometry

A

coutning cells by having them pass through a narrow tube and detecting them by a laser

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19
Q

lag phase

A

adaptation to prior cell division

line is horizontal, no increase in the number of bacteria

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20
Q

exponential (log) phase

A

rapid doubling

line has a positive slope

21
Q

stationary phase

A

occurs after the exponential log phase

line is horizontal

growth rate= death rate

22
Q

death phase

A

decline due to lack of food or other variable

negative slope

happens after stationary phase

23
Q

what does differential centrifugation separate

A

organelles and macromolecules based on size and density

24
Q

most dense to least dense organelles

A

nuclei
mitochondria/ chloroplasts
ER fragments
ribosomes

25
Q

karyotyping

A

observing chromosomes under light microscope during METAPHASE

used to diagnose conditions involving chromosomal abbherations, breakages, aneuploidies

26
Q

how is DNA sequencing done

A

dideoxy chain termination/sanger sequencing
next gen sequencing

both may use shotgun sequencing

27
Q

what is shotgun sequencing

A

cloned DNA genomes are cut into peices, sequenced, and recompiled to observe sequence overlaps

used by sanger sequencing/ dideoxy chain termination and next gen sequencing

28
Q

ddNTPs

A

dideoxynucleotides

used in sanger sequencing
lack two hydroxyl groups

with with normal dNTPs for DNA pol to use - if they are added to a DNA strand, itll terminate elongation bc there is no 3’ OH to form a new phosphodiester bond

29
Q

single nucleotide polymorphisms

A

serve as markers for disease causing genes in humans

30
Q

where do restriction enzymes cut DNA

A

at pallindromic sequences

31
Q

RFLPs

A

restriction fragment length polymorphisms

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

32
Q

exonucleases

A

result in sticky ends by cleaving nucleotides from the ends ofpolynucleotide chain

33
Q

endonucleases

A

cleave within a polynucleotide chain

result in either sticky or blunt ends

34
Q

how is DNA fingerprinting done

A

looking at peoples RFLPs or short tandem repeats STR

both unique stratches of DNA that allow for the identification of individuals

35
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 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.

36
Q

what are the steps of PCR

A

denaturation at 95 degrees to separate DNA strands

primer annealing at 65 degrees

elongation at 70 degrees - using Taq polymerase E

37
Q

what is used for gene therapy

A

virsues used to insert genes into cells bc they have the highest transduction efficiency- can cause immune response

can also use naked plasmid DNA and CRISPR- less efficient

38
Q

ESLISA

A

enzyme linked immunosorbent assay

determine if someone has a certain antigen to diagnose disease

Ab placed in microtiter plate with sample- change colour if antigen is present bc secondary antibody bound to colour with bind to the primary antibody

39
Q

pulse chase experiment

A

studies gene expressoin and fate of proteins- view how protein moves through a cell

pulse- aa radioactively labelled and incorporated into proteins

chase- prevent radioactively labelled protein production

protein tracked by simple staining

40
Q

gel electrophoresis

A

separate DNA by charge and size using electric field on an agarose gel

top- -ve cathode
bottom- +ve anode

smaller fragments travel farther to bottom

41
Q

SDS

A

sodium dodecyl sulfate

strong detergent used in gel electrophoersis to dentaure, linearize, and add neg 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

nothern 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).

45
Q

FRAP

A

flouresence recovery after photobleaching

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 over time due to cell dynamics
→ area gradually recovers
fluorescence.

46
Q

FLIM

A

flouresence lifetime imaging microscopy

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.

47
Q

genome annotation

A

identifies location of a gene and coding regions in genome

determines their functions

48
Q

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.

49
Q

DNA microarrays

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.

DAT BIOLOGY (32 decks)

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