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biological chem DAT > chp 8 BIO DAT > Flashcards

chp 8 BIO DAT Flashcards

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1
Q

overview of microscope

A
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2
Q

what is heat fixation ?

A

when FRESHLY HARVESTED cells are placed on a slide , then they are heated up with bunsen burner ( cells are glued to slide and killed ) then they are stained

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3
Q

why is fixation important?

A

because it preserves the cells most life like state and it holds the stain better

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4
Q

what is staining ?

A

adding color to the cells to emphasize cell structure. the staining process usually kills the cells

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5
Q

optical microscopy -

A

it involved shining a light on a sample and this method can be used to observe living cells

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6
Q

electron microscopy -

A

it shoots beams of electrons at a sample instead of light. it gives higher resolution images than optical microscopy. CANT be used to observe living cells.

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7
Q

if you want to observe a living cell what TYPE of microscope would you use ?

A

optical or electron ?

optical

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8
Q

what is resolution?

A

describes the ability of the microscope to distinguish detail

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9
Q

what is contrast ?

A

different in light and light intensity that makes the obj distinguishable

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10
Q

types of optical microscope : stereo micro-

A

offers low magnification to observe the surface of LIVE samples.
pro-observe living cells
con-resolution Is low

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11
Q

types of optical microscope : compound micro-

A

used to view ONE CELL THICK live samples.
-brightfield mi cro is a type of compound micro with a light to illuminate samples

pro-can observe single cell layers
con-poor contrast, may require staining

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12
Q

types of optical microscope : phase contrast micro-

A

can view thin samples of live cells with good contrast.
light passes through annular ring that creates phase shifts = good contrast

pro-detailed observation , good contrast and resolution

con-cant be used on thick samples and halo effects on samples edges

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13
Q

phase shift

A

slight difference in the positioning of the light

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14
Q

halo effect

A

halo effect is large a phase that surrounds the specimen

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15
Q

types of optical microscope : fluorescence micro-

A

uses a fluorescent substance to illuminate a specimen and observe its fluorescent components

pro-living samples , thin slices, can look at specific parts
con-fluorescence can cause artifacts

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16
Q

fluorophores

A

chemical that will light up when excited by another light source

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17
Q

dichroic filter

A

is used to allow specific wavelength of light to be reflected onto sample

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18
Q

types of optical microscope : confocal laser scanning micro-

A

frequently used with fluorescence tagging to observer chromosomes during mitosis. increases overall resolution but reduces intensity because of the screen

pro-overcomes artifacts. increasing resolution

con-light reduced and sample will need more light

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19
Q

confocal micro - can overcome what -

A

artifacts , by focusing beam of UV light at sample and has a screen with a small hole to block out of focus light from reaching detector

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20
Q

types of optical microscope : dark field micro-

A

able to view unstained samples of live cells by increasing contrast btw sample and background

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21
Q

how is contrast achieved with dark field micro-

A

by only allowing the light that goes through the sample and scatter to contact light detector. sample will appear one completely black background therefore low light intensity.

pro-excellent contrast on living Samples
cons -low light intensity

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22
Q

types of electron micro

A
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23
Q

electron micro-

A

electrons are shot up through a vacuum at a fixed and metal rated sample

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24
Q

vacuum -

A

prevents electrons from deviating their paths. (making them in a straight line)

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25
Q

fixation -

A

prevents proteins and structures from degrading

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26
Q

metal coat-

A

a stain using gold or palladium to coat the sample.

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27
Q

types of electron micro: scanning electron micro - SEM

A

high resolution 3D images of dehydrated samples surface. it captures electrons that scattered by atoms on the surface if the dehydrated sample

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28
Q

SEM pro and cons -

A

p- high 3D resolution of samples surface
c-costly , extensive sample prep-kills sample

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29
Q

types of electron micro: cryo scanning electron micro-

CRYO SEM

A

the sample is frozen in liquid nitrogen instead of dehydration.
this freezing provides 3D images of sample surface in its more natural form

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30
Q

CRYO SEM pros and cons

A

p-hight resolution and more natural form

c-extensive prep (kills sample) and artifacts due to freezing

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31
Q

types of electron micro: transmission electron microscopy
TEM

A

visualize hight resolution 2D images of a samples INTERNAL STRUCTURE

it captures the electrons that are transmitted through a thin slice of sample

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32
Q

TEM pros and cons

A

p-can view internal structures and hight resolution

c-costly and extensive prep

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33
Q

types of electron micro: electron tomography

A

provides 3D image of samples internal structure.
it sandwiches a bunch of TEM images together. (not considered a type pf microscopy )

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34
Q

electron tomography pros and cons

A

p-can look at obj and their relative position in 3D

c-costly and extensive prep

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35
Q

key terms —-

A
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36
Q

diplo

A

pairs

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37
Q

strep

A

chain along a single axis

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38
Q

staph

A

grape like clusters

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39
Q

coccus

A

spherical

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40
Q

bacillus

A

rod like

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41
Q

spiralla

A

spirals

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42
Q

hemocytometer

A

a gridded slide that samples are deposited on - manual counting of samples.

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43
Q

colony forming units

A

used to estimate the number of cells plate on a growth medium- assumption is made when each viable cell turns into visible colony

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44
Q

automated cell routing

A

cells show electrical resistance - mostly do not conduct electricity - estimation can be taken by observing flow of electricity in solution

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45
Q

bacterial growth curve -

A

lag, log,sationary,deeath

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46
Q

lag

A

bacteria are adapting to the environment- no growth

47
Q

log

A

“exponential phase” number of cells and rate growth doubles (linear increase)

48
Q

stationary

A

results from growth limitig factor . this is where growth=death (horizontal line)

49
Q

death

A

bacteria die due to lack of nutrients, temp , waste (declining )

50
Q

cell fractionation

A

process where cells contents are separated into fractions by centrifugation

51
Q

centrifuge

A

lab equipment that spins at very high speed- separates all the cell components by mass , density , or shape-

most dense and compact particles go to the bottom of centrifuge tube - pressed together as a pellet

52
Q

supernatant

A

whatever is not on the pellet-its the liquid surrounding

53
Q

centrifuge -

A

this method can also be used to separate proteins based on their solubility-
INSOUBLE -pellet
SOLUBLE-

54
Q

differential centrifugation- steps

A

the steps -
this is where cell care split open (HOMOGENIZATION) so components of the cells can be separated

55
Q

homogenate

A

mixture of split open cells from homogenization

56
Q

differential centrifugation-steps

A

homogenate is centrifuged
, nucleus will pellet first since its the most dense (everything else is the supernatant)

these steps are repeated

57
Q

density centrifugation

A

separates cell components original homogenate layers in a dingle centrifugation cycle.

results - cell components are arranged in layers

bottom layer - most dense

nuclei , mitochondria, chloroplast,ER fragments , ribosomes.

58
Q

why is stationary phase a horizontal line ?

A

bacterial growth is inhibited by growth factor , growth rate equals death rate

59
Q

karyotyping -

A

observation of chromosomes under a light microscope using staining.

shows number of chromes- and physical appearance

60
Q

karyotyping is a diagnostic tool for-

A

chromosomal aberrations(she and structure)

chromosomal breakage

extra or absence or chrome (chromosomal aneuploidy )

61
Q

in which phase of mitosis would karyotyping be performed ?

A

metaphase

62
Q

DAN sequencing –

A

idea : cutting along stretches of DNA into smaller pieces and sequence them

63
Q

2 types of sequencing –

A

sanger : (dideoxy chain termination ) old method

next gen : quicker , cheaper , common

64
Q

reasons for sequencing -

A

SNP: single nucleotide polymorphism
serves as a marker for genes that cause diseases.

65
Q

recombinant DNA–

A

produced when DNA frag from different sources are joined together

produced by restriction enzyme that cut DNA at palindromic sequences to make sticky or blunt ends

66
Q

palindromic sequences

A

inverted mirror of each other (RACE CAR)

67
Q

sticky ends and blunt ends

A

restriction enzymes cut at the palindromic sequence to make sticky and blunt ends.

68
Q

sticky ends -

A

HAVE unpaid nucleotides, makes it easier for complementary sticky ends to hybridize

69
Q

blunt ends -

A

DO NOT have unpaired nucleotides. harder to hybridize , less common

70
Q

CRISPR

A

an antiviral mechanism originally used by bacteria to target and remove sequences from invading pathogens ( scientist have adapted gene editing , where they select a region of genome and delete or add specific sequencing )

71
Q

PCR

A

process where million of pieces of DNA are copied automatically ( no cells required )

72
Q

requirements for PCR -

A

DNA primers
TAQ poly (heat resistant DNA poly)

73
Q

steps of PCR -

A

denaturation- 95C , container is heated , splits DNA double helix into separate single strands

primer annealing - 65C temp is lowered to allow DNA primers to stick to single DNA strand

elongation - 70C nucleotides added to 3’ end of dna using taq poly

74
Q

bacterial cloning -

A

a technique used to clone eukaryotic gene products in prokaryotic cells

75
Q

steps for bacterial cloning -

A

processed mRNA ( only exons) for eukaryotic gene of interest is located

mRNA is treated with reverse transcriptase to make complementary DNA

restriction enzyme and DNA ligase allow complementary DNA to be incorporated into plasmid

vector containing gene os taken up by competent bacterial cells (outside cells)

bacteria that took up vectors undergoes transformation (cell genome is changed by addition of DNA from outside )

check to see which has the antibiotic resistance or color change

76
Q

how to make competent bacterial cells -

A

competent bacteria is able to undergo transformation .
there are 2 types : electroporation and heat shock

77
Q

electroporation

A

electricity is applied to cells, this creates a temporary hole in the plasma membrane , the hole allows for transformation to occur

78
Q

heat shock

A

occurs in calcium chloride solution containing bacteria and vectors.
rapid increase in temp of solution creates hols in cell membrane. vectors are able to enter then transformation occurs

79
Q

checking for gene of interest -

A

transformation Is not always successful so we need to check if gene is successful with bacteria

80
Q

antibiotic resistance

A

gene that gives antibiotic resistance is attached to target gene, only cells with successful trans- will survive on the growth plate containing antibiotic

81
Q

color changing method

A

vectors contain gene that makes cell blue is used, if we target a gene successfully insert it into cell - blue gene is inactivated = cell turns white

if target gene is not inserted into cell , the cell will remain blue

82
Q

gel electrophoresis

A

separates DNA or RNA fragments by charge and size

83
Q

steps for gel electrophoresis -

A

DNA Is cut up by using restriction enzymes -

DNA is loaded into wells in the agarose gel

neg at TOP and pos at BOTTOM

electric filed I applied to the gel

neg charge DNA will be attracted to pos end of gel

pores of gel will obstruct movement of larger frag

84
Q

electrophoresis frag movement -

A

SMALLER frag of DNA/RNA the closer it will get to the bottom positive anode

LARGER frag will stay closer to the top neg anode

85
Q

southern blot

A

technique used to identify frag of a known DNA sequence in a large pop of DNA

86
Q

southern blot ; how it works

A

DNA frag are electrophoresed (separated by charge )

frag are separated into single stranded frag

single strands frag will be transferred from gel to membrane

membrane is washed with radio labeled DNA probes

87
Q

DNA probe -

A

fluorescent or radioactively labeled tool that identifies specific sequence within a large sample

88
Q

northern blot

A

same process but technique is used to identify fragments of a known RNA sequence using RNA probes

89
Q

western blot

A

same process but technique to quantify amount of target protein in sample .

achieved by using SDS PAGE ( sodium dodecyl sulfate polyacrylamide gel electrophoresis )

90
Q

SDS PAGE

A

used to separate proteins by mass

91
Q

SDS does what

A

denatures and gives a neg charge to the proteins

the neg charge proportions bettie the mass of the proteins because they go through the electrophoresis through the polyacrylamide gel

92
Q

proteins are treated with what -

A

primary and secondary antibodies

p - will selectively bind to target protein
s-will selectively bind to primary antibody

93
Q

ELISA ( enzyme linkend immunosorbent assay )

A

used to determine if a specific antigen exist in a person . ( aids in HIV )

94
Q

how ELISA works

A

blood is take, antibodies from that blood are placed into a microliter plate, if antibodies bind to antigen being tested for , there will be a color change on the plate. ( color change means antigen is present )

95
Q

pulse chase exp -

A

get to know more about how proteins move through cells

96
Q

how pulse chase works -

A

pulse - radioactive labeled amino acids are added to cell during short amount of time- the radioactive amino acids become part of the protein

chase - non radioactive aminos will be added to cell , this prevents every protein from being radioactively labeled.

can track progress though simple staining.

97
Q

genomics -

A

the study of all genes present in organisms

98
Q

genomic library , how it works -

A

restriction genes cut the genome into frag-

frag are then cloned using PCR

DNA ligase inserts the cloned frag into plasmids (plasmids preserves)

99
Q

DNA microarray -

A

is a chip that has thousands of DNA probes that are complementary to certain gene

100
Q

purpose of DNA microarray

A

determine which gene are expressed - fluorescence is used when hybridization occurs

101
Q

microarray steps -

A

isolate specific type of cell from sample - remove all the mRNA

reverse transcriptase synthesis from complementary DNA to mRNA

hybridize cDNA with DNA probe on microarray

use analysis machine ti examine microarray fro fluorescence

102
Q

transgenic animals

A

models that researches use to identify function of a gene - gene sequencing is taken from one organism and inserted into another though recombinant DNA tech

103
Q

repro cloning -

A

process taking a SOMATIC cell from animals and producing an exact genetic copy of that cell

104
Q

to clone animal

A

somatic cell needs to be reverted from mutipotent to totipotent

105
Q

cell potency

A

totipotent - has the ability to divide and produce entire org

pluripotent - can differentiate into any of the 3 germ layers , endoderm, mesoderm , ectoderm.
can’t make entire org

multipotent- most differentiated , can’t develop entire org

106
Q

chromatography -

A

separates liquids in mixture bu solubility

107
Q

components of chroma-

A

sample- sample is dissolved in solvent

mobile phase - the solvent itself
stationary phase - doesnt move

108
Q

fluorescence recovery after photobleaching

A

how and where biomolecules are moving in a living cell

109
Q

steps fro FRAP

A

measure baseline
area of sample is photobleach (pigmented molecules to permanently lose fluorescence )
photobleaching ,molecules are replaced by unbleached molecules over time
fluorescence area is restored

110
Q

fluorescence lifetime image microscopy

A

provides quantitative measure of concentration of various ion , molecules and gases in cell

111
Q

how FLIM works

A

achieved by irradiating cell samples with light and measuring their fluorescent lifetime (amount of time it takes cell to release fluorescence )

112
Q

concentration

A

(1/n)^#steps

113
Q

dilution

A

process of decreasing the concentration of solute in solution

1/concent-

biological chem DAT (12 decks)

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