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DAT BIO > DAT bio Chapter 8 Microscopes > Flashcards

DAT bio Chapter 8 Microscopes Flashcards

1
Q

What is fixation

A

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

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2
Q

2 types of fixation

A

heat and chemical

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3
Q

heat fixation

A

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

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4
Q

What does staining do

A

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

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5
Q

Optical microscopy

A

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

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6
Q

Electron microscopy:

A

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.

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7
Q

Requirement to see cells on electron microscope

A

cells must be fixed, stained, and killed

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8
Q

Stereo microscopes (

A

(dissection microscopes):

use low magnification to view the surface of an object

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9
Q

Compound microscopes

A

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

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10
Q

example of compound microscope

A

bright field microscopes

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11
Q

Bright field microscopes:

A

compound microscopes with a bright light

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12
Q

Phase contrast microscopes:

A

view: thin samples with live cells.

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

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13
Q

How does phase contrast microscopes achieve its high contrast?

A

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

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14
Q

How can you reduce halo effects in phase contrast microscopes?

A

can be reduced with

phase plates or thinner samples

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15
Q

Fluorescence microscopy

A

: 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.

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16
Q

Confocal laser scanning microscopy:

A

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

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17
Q

Confocal laser scanning microscopy was made to overcome _____

A

artifacts or distortions of fluorescence microscopy.

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18
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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19
Q

Scanning electron microscopy (SEM)

A

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

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20
Q

Cryo-scanning electron microscopy

A

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

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21
Q

Transmission electron microscopy (TEM)

A

high resolution 2D images of the sample’s

internal structures.

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22
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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23
Q

3 techniques to count cells

A

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

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24
Q

Hemocytometers

A

(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

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25
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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26
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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27
Q

Electrical resistance does what

A

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

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28
Q

Bacterial growth curve

A

describe the growth pattern of a given culture of cells

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29
Q

Bacterial growth curve has 4 stages

A
  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
30
Q

Cell fractionation

A

separates cell

contents by centrifugation

31
Q

what is centrifuge

A 
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.
32
Q

Differential centrifugation

A

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.

33
Q

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

A

homogenization

34
Q

What is supernatant

A

clear liquid that lies above the solid residue after centrifugation,

35
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

36
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)

37
Q

DNA sequencing

A

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

38
Q

2 methods for DNA sequencing

A

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

39
Q

When is recombinant DNA produced?

A

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).

40
Q

Restriction fragment length

polymorphisms (RFLPs) function

A

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?

41
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

42
Q

CRISPR

A

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

43
Q

Polymerase Chain Reaction (PCR):

A

automated

process creating millions of copies of DNA

44
Q

3 steps for 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.

45
Q

Bacterial cloning

A

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

46
Q

Steps (protocol for bacterial cloning) PART 1

A

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

47
Q

Steps (protocol for bacterial cloning) PART 2

A 
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)
48
Q

Steps (protocol for bacterial cloning) PART 3

A

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

49
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

50
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

51
Q

Northern blotting

A

identifying fragments of

known RNA using an RNA probe.

52
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).

53
Q

What is a DNA probe?

A

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

54
Q
  1. 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.

55
Q

Pulse chase experiments:

A

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

56
Q

Genomics

A

study of all genes present in an

organism’s genome and how they interact.

57
Q

genomic library

A

stores the DNA of an

organism’s genome

58
Q

DNA microarrays

A

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

59
Q

Protocol for DNA microarrays

A 
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
60
Q

Transgenic animals

A

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.

61
Q

Reproductive cloning:

A

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

62
Q

Reproductive cloning

- totipotent cells

A

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

63
Q

Pluripotent cells:

A

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.

64
Q

Multipotent cells

A

can give rise to some of

the three germ layers - not all. These cells are most differentiated. cannot develop entire organisms

65
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

66
Q

Fluorescence Recovery After

Photobleaching (FRAP):

A

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

67
Q

Fluorescence Recovery After

Photobleaching (FRAP): protocol

A 
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.
68
Q

2 types of live cell visualization that utilize fluorescence.

A

FRAP and FLIM

69
Q

Fluorescence Lifetime Imaging Microscopy

(FLIM):

A

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

70
Q

Knockout mice:

A

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

DAT BIO (25 decks)

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