Biology - Mircoscopy & Lab techniques

DAT biology Section

fixation; stained

_____ adheres cells to microscope slides in their most lifelike state, and it makes it easier for those cells to be _____

living cells are placed on a slide --> the slide is passed over a flame to kill/"glue" the cells to the slide --> stain is applied

describe the process of heat fixation

staining

_____ is the process of adding color to cells, which allows them to be viewed more easily under microscope

living

most optical microscopy techniques can be used to view _____ samples of cells

it involves shining light on a sample that reflects off of it and passes through lenses that magnify the object

how does optical microscopy work?

electron, optical

_____ microscopy allows for higher magnification than _____ microscopy

no, due to fixation and staining

can electron microscopy be used to look at living specimens? why or why not?

electron

most viruses are so small that they must be viewed using _____ microscopy.

it bombards a sample with electrons that bounce off the sample and pass through magnetic fields onto a screen. The image produced is viewed indirectly on a computer.

how does electron microscopy work?

they are light microscopes that can be used to view living samples

what is an advantage of stereo-microscopes (dissection microscopes)?

higher; smaller

electron microscopy offers _____ (higher/lower) resolution than optical microscopy because the wavelength of an electron is _____ (larger/smaller) than that of light

they are light microscopes that have a low resolution

what is a disadvantage of stereo-microscopes (dissection microscopes)?

compound microscopes

_____ are light microscopes that focus visible light to produce a 2D image of thin samples (single cell layers)

lens magnigifications

compound light microscopes usually have different _____, which gives them the ability to make more resolute images than a stereo-microscope (dissection light microscope)

they can be used to view 2D images of living samples (1 cell thick)

what is an advantage of compound light microscopes?

they only view samples that are 1 cell thick and they have a poor contrast, which means some samples may need to be fixed & stained (killed)

what are some disadvantages of compound light microscopes?

phase-contrast microscopes

_____ are optical microscopes that use light phase changesand contrast to produce 2D image of thin samples

good resolution and contrast; can be used to observe thin samples of living cells - including their internal structures

what are some advantages of phase-contrast optical microscopes?

ineffective on thick samples; halo effect around sample edges

what are some disadvantages of phase-contrast optical microscopes?

using phase plates to reduce the phase shift; use thinner samples

what are some strategies to reduce the halo effect of phase-contrast optical microscopy?

fluorophores

_____ are fluorescent chemicals that will re-emit light upon being excited by another light source

fluorescence

_____ is the emission of photons (light) from a particle that has absorbed light

fluorescence; confocal laser scanning

_____ and _____ are optical microscopy techniques that use laser light to produce 2D images of samples that have been tagged with fluorophores

colorful, 2D images of thin samples of living cells; increased brightness

what are some advantages of fluorescence optical microscopy?

fluorescence sometimes creates distortions (artifacts) that reduce the resolution

what are some disadvantages of fluorescence optical microscopy?

colorful, 2D images of thin samples of living cells; view chromosomes during mitosis; overcomes fluorescence artifacts (higher resolution)

what are some advantages of confocal laser scanning optical microscopy?

reduced light intensity and longer illumination times than fluorescence optical microscopy

what are some disadvantages of confocal laser scanning optical microscopy?

dark field optical microscopy

in _____, only scattered light from the sample is transmitted to produce 2D images of unstained, living cells

excellent contrast on living samples of unstained cells (black background)

what is an advantage of dark field optical microscopy?

low light intensity

what is a disadvantage of dark field optical microscopy?

vacuum (the vacuum prevents electrons from deviating in path)

in electron microscopy, electrons are shot through a _____ at a sample which has been fixed and
metal coated (cells are dead)

scanning electron microscopy (SEM)

_____ captures electrons that are scattered by atoms found on the surface of dehydrated samples

high resolution, 3D images of sample surfaces

what is an advantage of SEM?

it is costly, and the fixation/staining/dehydration kills the sample

what are some disadvantages of SEM?

cryo-scanning electron microscopy (cryo-SEM)

_____ is like SEM, but the sample is frozen instead of dehydrated

high resolution, 3D images of sample surfaces, which are presented in a more natural form than SEM (due to freezing)

what are some advantages of cryo-SEM?

it is costly, and the fixation/staining/freezing kills the sample

what are some disadvantages of cryo-SEM?

transmission electron microscopy (TEM)

_____ captures electrons that are transmitted through a thin slice of a sample

high resolution 2D images of internal sample structures

what are some advantages of TEM?

it is costly, and the extensive sample preparation kills all living cells

what are some disadvantages of TEM?

electron tomography (not a form of mircoscopy)

_____ integrates multiple TEM 2D images into a 3D model

can look at objects and their relative positions in 3D

what are some advantages of electron tomography?

it is costly, and the extensive sample preparation kills all living cells

(because it is based on TEM)

what are some disadvantages of electron tomography?

SEM

is SEM or TEM used to look at surfaces?

cell counting chambers

what are hemocytometers?

colony forming units (CFUs)

_____ are used to estimate the number of cells plated on a growth medium

colony

colony forming units (CFUs) are based on the assumption that each viable cell initially plated gave rise to a _____

electrical resistance and flow cytometry

what are 2 methods for automated cell counting?

number of cells

as cells show electrical resistance and impede conductance, the _____ in a solution can be estimated by observing the flow of electricity

flow cytometry

in _____, cells pass through a very narrow tube and can be counted via detection by a laser beam.

cell fractionation

_____ is the process where cell contents are separated into their fractions (one part of a whole) by centrifugation

centrifuge

a _____ is a laboratory apparatus that spins in a circular path at very high speeds.

mass, density, and/or shape

centrifugation separates cell components through _____

sediment; pellet

(top liquid is the supernatant)

in centrifugation, the densest and most compact particles will _____ to the bottom of the tube first, becoming pressed together as a _____ (precipitate)

proteins

(insoluble proteins pellet out, while the soluble proteins remain in the supernatant)

centrifugation can be used to separate _____ based on solubility

differential centrifugation (the homogeneous is centrifuged/fractionated)

in _____, cells are split open with a blender and the resulting homogenate is separated based on mass, density, and/or shape

density

_____ centrifugation separates cell contents in just 1 spin step, creating multiple layers separated by density

nuclei > mitochondria/chloroplast > ER fragments > ribosomes

arrange the following organelles from most to least dense: endoplasmic reticulum (ER), ribosomes, mitochondria, nuclei, chloroplasts

Karyotyping

_____ is the observation of chromosomes under a light microscope using staining

number; physical appearance

a karyotype shows both the _____ of chromosomes and their _____

metaphase

karyotyping is preformed during _____

Down syndrome (or trisomy 21); karyotyping

_____ is a condition that results in a third copy of chromosome 21, and _____ allows for substantiation of its diagnosis

single nucleotide polymorphisms (SNPs)

for the most part, the human genome is the same, with slight differences in the sequence every ~ 1000 nucleotides (called _____), which serve as markers for genes that cause disease

dideoxy chain termination (Sanger sequencing) and next generation sequencing

what are the 2 most common methods for DNA sequencing?

dideoxy chain termination (Sanger sequencing); next generation sequencing

_____ is an older and more established method of DNA sequencing, while _____ is used more often now because it is quicker and cheaper

recombinant DNA

_____ is produced when DNA fragments from different sources are joined together

palindromic sequence

a _____ occurs when there is a block of nucleotides that are inverted mirrors of each other

restriction enzymes; sticky or blunt

the DNA fragments that get incorporated into recombinant DNA are produced by _____, which tend to cut DNA at palindromic sequences to produce _____ ends

unpaired nucleotides

(complimentary sticky ends are made by the same restriction enzyme)

sticky ends have _____, which makes it easy for complementary sticky ends to hybridize

blunt ends (blunt ends are harder to hybridize because of the paired nucleotides)

_____ are less common than sticky ends, and they do not have unpaired nucleotides

restriction fragment length polymorphisms (RFLPs)

_____ are unique lengths of DNA that result from restriction enzymes, allowing for the comparison between individuals

short tandem repeats (STRs)

a _____ is a group of nucleotides that repeats multiple times in a stretch of DNA

twins

in which individuals are RFLPs and STRs not unique?

DNA fingerprinting

RFLPs and STRs are used in _____, which is a technique that may be used in paternity and forensic cases

Polymerase chain reactions (PCR)

the _____ is an automated biotechnology process that can quickly create millions of copies of DNA, and it requires no cells

DNA to be cloned; nucleotides; DNA primers; heat-resistant DNA polymerase (Taq polymerase)

PCR can be carried out in a single container - list the components that container needs to contain in order for PCR to take place:

denaturation; primer annealing; elongation

what are the 3 cyclical steps of PCR?

eukaryotic gene products; prokaryotic cells

(note that the insulin gene is obtained as cDNA from processed human mRNA)

bacterial cloning is an important technique to produce medicines because _____ are cloned in _____

processed mRNA

_____ corresponds to a eukaryotic gene with all introns removed (used in microarrays and bacterial cloning)

complementary DNA (cDNA)

_____ is DNA made from RNA, and it is used in microarrays and bacterial cloning

reverse transcriptase

_____ produces complementary DNA (cDNA) from mRNA, and it is relied upon for microarrays and bacterial cloning

DNA ligase

_____ catalyzes phosphodiester bonds between the ends of DNA restriction fragments (used heavily in genomic libraries and bacterial cloning)

plasmids

_____ are circular pieces of extrachromosomal DNA in bacteria (used in bacterial cloning and genomic libraries)

vector (used heavily in genomic libraries and bacterial cloning)

a _____ is a piece of DNA (such as a plasmid) that can be taken up by competent cells

_____ is a process that occurs when a cell's genome is changed by the addition of DNA that was once floating freely in the environment

transformation

transformation; electroporation

competent bacterial cells can undergo _____, and they can be made competent through _____

electroporation

_____ is a process where electricity is applied to cells, creating temporary holes in the plasma membrane

antibiotic resistance and color change

what are 2 key methods for selecting bacterial cells that have undergone transformation in bacterial cloning?

charge; size

gel electrophoresis separates macromolecular fragments on their _____ and _____

negative cathode at the top; positive anode at the bottom

gel electrophoresis has a _____ (positive/negative) _____ (anode/cathode) at the top and a _____ (positive/negative) _____ (anode/cathode) at the bottom

smallest

in gel electrophoresis, the _____ (smallest/largest) fragments travel the furthest

probe

a _____ is a fluorescent or radioactively labeled tool that allows scientists to identify a specific sequence within a large sample

Southern blotting; DNA

_____ is an electrophoresis technique for separating DNA fragments, and it uses _____ probes

Nothern blotting; RNA

_____ is an electrophoresis technique for separating RNA fragments, and it uses _____ probes

western blotting; primary and secondary antibodies

_____ is an electrophoresis technique for separating proteins, and it uses _____ as the probes

agarose; SDS-PAGE

southern and northern blotting tend to use _____ gel, whereas western blotting tends to use _____

enzyme-linked immunosorbent assay (ELISA)

_____ is a technology to determine if a specific antigenexists in a person, aiding in the diagnoses/exposure to certain diseases

antibodies; antigens

ELISA is based on the idea that a person will have _____ for a given disease's _____ if they have the disease, or have been exposed to it

Pulse Chase

_____ experiments allow for the visualization/tracking of molecules of interest throughout a cell

add radioactive amino acids during the pulse —→ radioactive protein synthesis —→ wash away radioactive amino acids —→ add regular amino acids during the chase

describe the general idea of a pulse chase experiment for proteins: