chp 8 BIO DAT Flashcards
overview of microscope
what is heat fixation ?
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
why is fixation important?
because it preserves the cells most life like state and it holds the stain better
what is staining ?
adding color to the cells to emphasize cell structure. the staining process usually kills the cells
optical microscopy -
it involved shining a light on a sample and this method can be used to observe living cells
electron microscopy -
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.
if you want to observe a living cell what TYPE of microscope would you use ?
optical or electron ?
optical
what is resolution?
describes the ability of the microscope to distinguish detail
what is contrast ?
different in light and light intensity that makes the obj distinguishable
types of optical microscope : stereo micro-
offers low magnification to observe the surface of LIVE samples.
pro-observe living cells
con-resolution Is low
types of optical microscope : compound micro-
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
types of optical microscope : phase contrast micro-
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
phase shift
slight difference in the positioning of the light
halo effect
halo effect is large a phase that surrounds the specimen
types of optical microscope : fluorescence micro-
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
fluorophores
chemical that will light up when excited by another light source
dichroic filter
is used to allow specific wavelength of light to be reflected onto sample
types of optical microscope : confocal laser scanning micro-
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
confocal micro - can overcome what -
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
types of optical microscope : dark field micro-
able to view unstained samples of live cells by increasing contrast btw sample and background
how is contrast achieved with dark field micro-
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
types of electron micro
electron micro-
electrons are shot up through a vacuum at a fixed and metal rated sample
vacuum -
prevents electrons from deviating their paths. (making them in a straight line)
fixation -
prevents proteins and structures from degrading
metal coat-
a stain using gold or palladium to coat the sample.
types of electron micro: scanning electron micro - SEM
high resolution 3D images of dehydrated samples surface. it captures electrons that scattered by atoms on the surface if the dehydrated sample
SEM pro and cons -
p- high 3D resolution of samples surface
c-costly , extensive sample prep-kills sample
types of electron micro: cryo scanning electron micro-
CRYO SEM
the sample is frozen in liquid nitrogen instead of dehydration.
this freezing provides 3D images of sample surface in its more natural form
CRYO SEM pros and cons
p-hight resolution and more natural form
c-extensive prep (kills sample) and artifacts due to freezing
types of electron micro: transmission electron microscopy
TEM
visualize hight resolution 2D images of a samples INTERNAL STRUCTURE
it captures the electrons that are transmitted through a thin slice of sample
TEM pros and cons
p-can view internal structures and hight resolution
c-costly and extensive prep
types of electron micro: electron tomography
provides 3D image of samples internal structure.
it sandwiches a bunch of TEM images together. (not considered a type pf microscopy )
electron tomography pros and cons
p-can look at obj and their relative position in 3D
c-costly and extensive prep
key terms —-
diplo
pairs
strep
chain along a single axis
staph
grape like clusters
coccus
spherical
bacillus
rod like
spiralla
spirals
hemocytometer
a gridded slide that samples are deposited on - manual counting of samples.
colony forming units
used to estimate the number of cells plate on a growth medium- assumption is made when each viable cell turns into visible colony
automated cell routing
cells show electrical resistance - mostly do not conduct electricity - estimation can be taken by observing flow of electricity in solution
bacterial growth curve -
lag, log,sationary,deeath
lag
bacteria are adapting to the environment- no growth
log
“exponential phase” number of cells and rate growth doubles (linear increase)
stationary
results from growth limitig factor . this is where growth=death (horizontal line)
death
bacteria die due to lack of nutrients, temp , waste (declining )
cell fractionation
process where cells contents are separated into fractions by centrifugation
centrifuge
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
supernatant
whatever is not on the pellet-its the liquid surrounding
centrifuge -
this method can also be used to separate proteins based on their solubility-
INSOUBLE -pellet
SOLUBLE-
differential centrifugation- steps
the steps -
this is where cell care split open (HOMOGENIZATION) so components of the cells can be separated
homogenate
mixture of split open cells from homogenization
differential centrifugation-steps
homogenate is centrifuged
, nucleus will pellet first since its the most dense (everything else is the supernatant)
these steps are repeated
density centrifugation
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.
why is stationary phase a horizontal line ?
bacterial growth is inhibited by growth factor , growth rate equals death rate
karyotyping -
observation of chromosomes under a light microscope using staining.
shows number of chromes- and physical appearance
karyotyping is a diagnostic tool for-
chromosomal aberrations(she and structure)
chromosomal breakage
extra or absence or chrome (chromosomal aneuploidy )
in which phase of mitosis would karyotyping be performed ?
metaphase
DAN sequencing –
idea : cutting along stretches of DNA into smaller pieces and sequence them
2 types of sequencing –
sanger : (dideoxy chain termination ) old method
next gen : quicker , cheaper , common
reasons for sequencing -
SNP: single nucleotide polymorphism
serves as a marker for genes that cause diseases.
recombinant DNA–
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
palindromic sequences
inverted mirror of each other (RACE CAR)
sticky ends and blunt ends
restriction enzymes cut at the palindromic sequence to make sticky and blunt ends.
sticky ends -
HAVE unpaid nucleotides, makes it easier for complementary sticky ends to hybridize
blunt ends -
DO NOT have unpaired nucleotides. harder to hybridize , less common
CRISPR
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 )
PCR
process where million of pieces of DNA are copied automatically ( no cells required )
requirements for PCR -
DNA primers
TAQ poly (heat resistant DNA poly)
steps of PCR -
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
bacterial cloning -
a technique used to clone eukaryotic gene products in prokaryotic cells
steps for bacterial cloning -
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
how to make competent bacterial cells -
competent bacteria is able to undergo transformation .
there are 2 types : electroporation and heat shock
electroporation
electricity is applied to cells, this creates a temporary hole in the plasma membrane , the hole allows for transformation to occur
heat shock
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
checking for gene of interest -
transformation Is not always successful so we need to check if gene is successful with bacteria
antibiotic resistance
gene that gives antibiotic resistance is attached to target gene, only cells with successful trans- will survive on the growth plate containing antibiotic
color changing method
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
gel electrophoresis
separates DNA or RNA fragments by charge and size
steps for gel electrophoresis -
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
electrophoresis frag movement -
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
southern blot
technique used to identify frag of a known DNA sequence in a large pop of DNA
southern blot ; how it works
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
DNA probe -
fluorescent or radioactively labeled tool that identifies specific sequence within a large sample
northern blot
same process but technique is used to identify fragments of a known RNA sequence using RNA probes
western blot
same process but technique to quantify amount of target protein in sample .
achieved by using SDS PAGE ( sodium dodecyl sulfate polyacrylamide gel electrophoresis )
SDS PAGE
used to separate proteins by mass
SDS does what
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
proteins are treated with what -
primary and secondary antibodies
p - will selectively bind to target protein
s-will selectively bind to primary antibody
ELISA ( enzyme linkend immunosorbent assay )
used to determine if a specific antigen exist in a person . ( aids in HIV )
how ELISA works
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 )
pulse chase exp -
get to know more about how proteins move through cells
how pulse chase works -
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.
genomics -
the study of all genes present in organisms
genomic library , how it works -
restriction genes cut the genome into frag-
frag are then cloned using PCR
DNA ligase inserts the cloned frag into plasmids (plasmids preserves)
DNA microarray -
is a chip that has thousands of DNA probes that are complementary to certain gene
purpose of DNA microarray
determine which gene are expressed - fluorescence is used when hybridization occurs
microarray steps -
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
transgenic animals
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
repro cloning -
process taking a SOMATIC cell from animals and producing an exact genetic copy of that cell
to clone animal
somatic cell needs to be reverted from mutipotent to totipotent
cell potency
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
chromatography -
separates liquids in mixture bu solubility
components of chroma-
sample- sample is dissolved in solvent
mobile phase - the solvent itself
stationary phase - doesnt move
fluorescence recovery after photobleaching
how and where biomolecules are moving in a living cell
steps fro FRAP
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
fluorescence lifetime image microscopy
provides quantitative measure of concentration of various ion , molecules and gases in cell
how FLIM works
achieved by irradiating cell samples with light and measuring their fluorescent lifetime (amount of time it takes cell to release fluorescence )
concentration
(1/n)^#steps
dilution
process of decreasing the concentration of solute in solution
1/concent-
