Bootcamp.com - Microscopy and Lab Techniques

https://quizlet.com/303771481/bootcampcom-microscopy-and-lab-techniques-flash-cards/

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

fixation; stained

describe the process of heat fixation

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

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

staining

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

living

how does optical microscopy work?

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

_____ microscopy allows for higher magnification than _____ microscopy

electron, optical

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

no, due to fixation and staining

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

electron

how does electron microscopy work?

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.

_____ are light microscopes that focus visible light to produce a 2D image of a sample's surface

stereo-microscopes (dissection microscopes)

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

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

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

higher; smaller

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

they are light microscopes that have a low resolution

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

compound microscopes

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

lens magnifications

what is an advantage of compound light microscopes?

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

what are some disadvantages 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)

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

phase-contrast microscopes

what are some advantages of phase-contrast optical microscopes?

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

what are some disadvantages of phase-contrast optical microscopes?

ineffective on thick samples; halo effect around sample edges

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

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

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

fluorophores

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

fluorescence

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

fluorescence; confocal laser scanning

what are some advantages of fluorescence optical microscopy?

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

what are some disadvantages of fluorescence optical microscopy?

fluorescence sometimes creates distortions (artifacts) that reduce the resolution

what are some advantages of confocal laser scanning optical microscopy?

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

what are some disadvantages of confocal laser scanning optical microscopy?

reduced light intensity and longer illumination times than fluorescence optical microscopy

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

dark field optical microscopy

what is an advantage of dark field optical microscopy?

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

what is a disadvantage of dark field optical microscopy?

low light intensity

in electron microscopy, electrons are shot through a _____ at a sample which has been fixed and

metal coated (cells are dead)

vacuum

(the vacuum prevents electrons from deviating in path)

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

scanning electron microscopy (SEM)

what is an advantage of SEM?

high resolution, 3D images of sample surfaces

what are some disadvantages of SEM?

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

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

cryo-scanning electron microscopy (cryo-SEM)

what are some advantages of cryo-SEM?

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

what are some disadvantages of cryo-SEM?

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

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

transmission electron microscopy (TEM)

what are some advantages of TEM?

high resolution 2D images of internal sample structures

what are some disadvantages of TEM?

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

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

electron tomography

(not a form of microscopy)

what are some advantages of electron tomography?

can look at objects and their relative positions in 3D

what are some disadvantages of electron tomography?

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

(because it is based on TEM)

is SEM or TEM used to look at surfaces?

SEM

what are hemocytometers?

cell counting chambers

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

colony forming units (CFUs)

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

colony

what are 2 methods for automated cell counting?

electrical resistance and flow cytometry

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

number of cells

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

flow cytometry

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

cell fractionation

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

centrifuge

centrifugation separates cell components through _____

mass, density, and/or shape

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

sediment; pellet

(top liquid is the supernatant)

centrifugation can be used to separate _____ based on solubility

proteins

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

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

differential centrifugation

(the homogenate is centrifuged/fractionated)

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

density

e.g. blood centrifugation

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

nuclei > mitochondria/chloroplast > ER fragments >

ribosomes

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

karyotyping

a karyotype shows both the _____ of chromosomes and their _____

number; physical appearance

karyotyping is preformed during _____

metaphase

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

Down syndrome (or trisomy 21); karyotyping

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

single nucleotide polymorphisms (SNPs)

what are the 2 most common methods for DNA sequencing?

dideoxy chain termination (Sanger sequencing) and 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

dideoxy chain termination (Sanger sequencing); next generation sequencing

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

recombinant DNA

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

palindromic sequence

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

restriction enzymes; sticky or blunt

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

unpaired nucleotides

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

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

blunt ends

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

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

restriction fragment length polymorphisms (RFLPs)

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

short tandem repeats (STRs)

in which individuals are RFLPs and STRs not unique?

twins

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

DNA fingerprinting

the _____ is an automated biotechnology process that can quickly create millions of

copies of DNA, and it requires no cells

polymerase chain reaction (PCR)

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

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

what are the 3 cyclical steps of PCR?

denaturation; primer annealing; elongation

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

eukaryotic gene products; prokaryotic cells

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

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

processed mRNA

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

complementary DNA (cDNA)

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

reverse transcriptase

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

DNA ligase

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

plasmids

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

vector

(used heavily in genomic libraries and bacterial cloning)

_____ 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

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

transformation; electroporation

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

electroporation

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

antibiotic resistance and color change

gel electrophoresis separates macromolecular fragments on their _____ and _____

charge; size

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

negative cathode at the top; positive anode at the bottom

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

smallest

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

probe

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

southern blotting; DNA

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

northern blotting; RNA

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

western blotting; primary and secondary antibodies

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

agarose; SDS-PAGE

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

enzyme-linked immunosorbent assay (ELISA)

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

antibodies; antigens

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

pulse chase