Upgrade to remove ads
DAT Chap 8
Terms in this set (74)
is the process of getting cells to 'stick' on a microscope slide, such that the cell is preserved in its most life-like state. Another benefit of cell fixation is that it makes it easier for cells to hold onto any stain that is applied.
First, freshly harvested cells will be placed on one side of a microscope slide. Then, the underside of that same slide will be passed over a bunsen burner flame. This causes the slide to heat-up, which 'glues' the cells on the slide. Heat fixation also preserves cells because the heat causes cell processes to stop - i.e. it kills them.
process of adding color to cells, such that researchers will have an easier time in seeing cell structures. However, staining is usually associated with cells that have been killed, either through the preceding fixation or the staining process itself. Many types of staining protocols will kill any living cells because alcohol is often used as a 'wash' for removing excess stain.
shining light on a sample. Then, the light that reflects (bounces) off the sample passes through a series of lenses, which magnify the object. The final image is observed by looking directly into the optical lens of the microscope. Many optical microscopy techniques can be used to observe living cells.
similar to optical microscopy. However, it bombards a sample with electrons rather than light. The electrons that bounce off the sample pass through a series of magnetic fields and ultimately land on a screen. From here, the image can be viewed indirectly, using a computer.
Requires fixed, stained , and killed
another name for dissection microscopes, which only offer low
magnification to observe the surface of live specimens.
microscope that can be used to view simple, one-cell thick, live cells. They have more than one lens, and each lens magnifies the sample by a set amount.
some living sample
may require staining
Bright field microscopes
compound microscopes that have a bright light to illuminate the sample (i.e. a bright field).
Phase contrast microscopes
visualization of thin samples containing live cells. Cells do not have to be fixed, stained, or tagged because phase contrast microscopes have tremendous contrast.
Light passes through an annular ring (which forms a cone of light), hits the object, and refracts when it passes through materials in the object with different densities. This changes the speed of the light, causing it to bend (refract). The difference between the refracted light (through the object) and unrefracted light (not through the object) creates a phase shift in the light.
detailed observation of living organism
ineffective thick samples
subtle difference in the positioning of light, detected by the phase contrast microscope. This creates tremendous contrast, and the microscope can compute what it is looking at by recompiling the image.
Sometimes, the area around the specimen is distorted by large phase shifts. This is known as the halo effect, and it can be reduced by using phase plates to reduce the phase shift. Another strategy to reduce the halo effect includes using thinner samples.
fluorescence microscopy, fluorophores
(a 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.
emission of photons (light) from a particle that has absorbed light. When a fluorophore absorbs light, electrons are excited to a higher energy level. When the electrons fall back down to their normal energy level, they release energy in the form of light.
fluorescence used in fluorescence microscopy creates distortions in the image.
decrease the overall resolution. They are created by fluorescent microscopes because they illuminate the entire specimen at one time. This causes the specimen's fluorophores to be excited simultaneously. While this helps to increase the brightness of the sample, it causes the background to be unfocused.
Confocal laser scanning microscopy
technique can be used without fluorescence tagging, it is more frequently used with fluorescence tagging to observe chromosomes during mitosis.
This type of microscopy was introduced to overcome the artifacts of fluorescence microscopy. It achieves this by focusing a beam of ultraviolet light at a sample. It also uses a screen with a small hole to prevent out-of-focus light from reaching the detector.
Dark field microscopy
optical microscopy technique that allows researchers to view unstained samples of live cells.
This is achieved by increasing the contrast between the sample and the field around the sample.
Light enters from the bottom of the microscope. The dark field patch stop blocks light from the center from entering the object; which creates an outer ring of light. The condenser lens refocuses this light back onto the sample.
Contrast is created by allowing only the light that passes through the sample and scatters to contact the light detector - all other light is blocked (including light that is directly transmitted through the sample). Only scattered light from the sample is transmitted.
This means that the sample image will appear on a completely black background. As a result of this, the light intensity can be low.
low light intensity
prevents the electrons from deviating their path.
prevents proteins and structures from degrading.
also be referred to as the stain, and it uses gold or palladium to coat the sample
Scanning electron microscopy (SEM)
captures electrons that are scattered by atoms found on the surface of dehydrated samples. For that reason, it allows researchers to visualize high resolution 3D images of the sample surface
looks at surface
Cryo-scanning electron microscopy
specific type of scanning electron microscopy where the sample is frozen in liquid nitrogen (cryogenic) instead of being dehydrated. This freezing process provides for a 3D image of the sample surface in its more natural form; however, it can sometimes create artifacts.
Transmission electron microscopy (TEM)
captures electrons that are transmitted through a thin slice of a sample. This allows researchers to view high resolution 2D images of a sample's internal structures. Like all types of electron microscopy, it is costly.
3D image of a sample's internal structures. This is achieved by sandwiching a bunch of TEM images together. For this reason, it is not considered a type of microscopy.
more simply as counting chambers - are a gridded slide upon which a sample is deposited. Under a microscope, the grid is used to manually count the number of cells in a known area. Then, the sample count is extrapolated for the full volume of the sample
Colony forming units (CFUs)
used to estimate the number of cells plated on a growth medium. This is with the assumption that each viable cell initially plated gave rise to a colony, which can be visibly seen and manually accounted for.
Automated cell counting
electrical resistance. As cells show electrical resistance and impede conductance, the number of cells in a solution can be estimated by observing the flow of electricity. In flow cytometry, cells pass through a very narrow tube and can be counted via detection by a laser beam.
process where cell contents are separated into their fractions (one part of a whole) by centrifugation. A centrifuge is a laboratory apparatus that spins in a circular path at very high speeds. This separates the cell components through their mass, density, and/or shape.
The densest and most compact particles will sediment to the bottom of the centrifuge tube first, becoming pressed together as a pellet (aka precipitate). Whatever is not in the pellet remains as a supernatant liquid on top of the pellet.
mixture of the split open cells produced by homogenization. It is stored in an inert buffer to preserve the cell components.
When the homogenate is centrifuged (spun), the nucleus will pellet first because it is the most dense. Everything else will remain as the supernatant. The supernatant will then be poured into a new centrifuge tube so the next most dense organelle can pellet. This process is repeated over and over to gradually fractionate (isolate) the cell components so they can be studied.
separates all the cell components of the original homogenate over the course of a single centrifugation cycle. Density centrifugation sediments the cell components into layers, with the most dense layers being found toward the bottom of the tube.
From most dense to least dense: nuclei > mitochondria/chloroplast > ER fragments > ribosomes.
under a light microscope using staining. A
karyotype shows both the number of chromosomes and their physical appearance.
Karyotyping is performed during metaphase of mitosis and can be used as a diagnostic tool
for multiple conditions involving chromosomal aberrations, breakages, and aneuploidies.
For example, Down syndrome (or trisomy 21) is a condition that results in a third copy of chromosome 21, karyotyping allows for substantiation of its diagnosis.
technology to sequence the nucleotides (adenine, thymine, guanine, and cytosine) in fragments of DNA. This is valuable because it allows researchers to determine the sequence of long stretches of DNA, simply by cutting them into smaller fragments. If this process is repeated for each chromosome in a given organism, the entire genome can be sequenced.
For the most part, the human genome is the same in all people. However, there are slight differences in the sequence every ~ 1000 nucleotides. These differences are called single nucleotide polymorphisms (SNPs), and they serve as markers for genes that cause disease.
dideoxy chain termination (Sanger sequencing) and next generation sequencing.
specifics of how these methods work is pretty low yield for the DAT, so we won't cover them in great detail
produced when DNA fragments from different sources are joined together. These fragments are produced by restriction enzymes, which tend to cut DNA at palindromic sequences to produce sticky or blunt ends.
when there is a block of nucleotides that are inverted mirrors of each other. For example, EcoRI is a restriction enzyme made by E. Coli.
unpaired nucleotides, which makes it easy for complementary sticky ends to hybridize. An important point to note is that complementary sticky ends are made from the same restriction enzyme. Restriction enzymes make these in the following way:
do not have unpaired nucleotides, which makes them harder to hybridize with other blunt ends. They are less common than sticky ends,
Restriction fragment length polymorphisms
unique lengths of DNA that result from restriction enzymes. This allows for comparison between individuals through the polymorphisms in DNA length.
The DNA that is analyzed in RFLPs is the non-coding DNA. Coding DNA (DNA that codes for genes/proteins) is highly conserved among humans.
DNA fingerprinting i
technique that may be used in paternity and forensic cases. This is because it identifies individuals through aspects of unique DNA, including RFLPs and short tandem repeats (STR's). An STR is a group of nucleotides that repeats again and again in a stretch of DNA.
RFLPs form a genomic 'fingerprint,' as every individual will have different length RFLPs after restriction enzymes are applied. The one exception is identical twins, which have the same DNA code and therefore the same RFLPs.
Since the number of STR's tends to vary significantly in the population, the DNA of an individual (e.g. a suspect in a crime) can be compared to the DNA of a sample (e.g. blood left at the scene of a crime) for a positive match.
Polymerase chain reaction (PCR)
biotechnology process that can quickly create millions of copies of DNA. PCR is automated, requiring no cells.
Before the actual PCR process is carried out, the reaction needs to be set up in a single container. The DNA to be cloned, RNA primers, and a heat resistant DNA polymerase (Taq polymerase) are added to the container.
Then, PCR can proceed by cycling between the following three steps:
1. Denaturation (~ 95 oC): the container is heated. This splits the DNA double helix into separate single strands.
2. Primer annealing (~ 65 oC): the temperature is slightly lowered. This allows RNA primers hybridize to the single strands of DNA.
3. Elongation (~ 70 oC): nucleotides (adenine, thymine, guanine, cytosine) are added to the 3' end of the RNA primer using Taq polymerase. Taq polymerase is a special heat stable DNA polymerase captured from thermophilic bacteria. It uses the single strand DNA fragments as a template.
1. Processed mRNA for the eukaryotic gene of interest is isolated.
2. The processed mRNA are treated with reverse transcriptase to make cDNA
3. A restriction enzyme and DNA ligase allow the cDNA to be incorporated into a plasmid,
which acts as the transfer vector in bacterial cloning.
4. The vector containing the gene is taken-up by competent bacterial cells.
5. Bacteria that have taken up the vector will undergo transformation.
6. We finding the gene of interest by using antibiotic resistance or color change methods.
Glossary for bacterial cloning:
corresponds to eukaryotic gene all introns remove.
works in "reverse" by transcribing RNA into cDNA.
Complementary DNA (cDNA)
complementary to the RNA it was made from.
inserts the cloned fragments into plasmids by catalyzing phosphodiester bonds between their ends.
circular pieces of extrachromosomal DNA in bacteria.
piece of DNA that can be taken up by competent cells. This allows the DNA to be replicated, transcribed into mRNA, and translated into protein.
cells can undergo transformation, and they can be made competent through electroporation.
process where electricity is applied to cells. This creates temporary holes in the plasma membrane, which allows transformation to occur.
process that occurs when a cell's genome is changed by the addition of DNA that was once floating freely in the environment.
Antibiotic resistance method:
Using restriction enzymes, a gene that confers antibiotic resistance is attached to the target gene. Only the cells that have picked up and integrated the antibiotic resistance gene/target combo will have antibiotic resistance. These cells will be the only ones that grow on a plate containing an antibiotic. Cells that did not pick up this combo will die because they are not resistant to the antibiotic.
Color change method:
Vectors containing a gene that make cells blue will be used. Restriction enzymes that cut the blue-gene will also be used. If the target gene inserts into the blue-gene, the blue-gene will be inactivated and the cell will appear white. If the target gene does not insert into the blue-gene, the gene will re-attach, remaining active and blue.
In the process of DNA gel electrophoresis, the DNA is first cut up into pieces using a restriction enzyme. It is then loaded into wells in the agarose gel. Recall that DNA is negatively charged due to the phosphate groups it contains.
Under the influence of the electrical field, the negatively charged DNA will want to sieve its way through the porous gel toward the positive anode at the bottom. However, the pores of the gel will obstruct the movement of larger fragments. Therefore, the smaller the fragment of DNA, the further it will travel away from the negative cathode at the top.
For this reason, gel electrophoresis separates fragments on their charge and size. If multiple samples are loaded, they can be compared to determine genetic similarities and differences.
technique to identify fragments of a known DNA sequence in a large population of DNA. After DNA fragments have been electrophoresed, they can be separated into single stranded fragments using a basic solution. These single stranded fragments will be transferred from the gel onto a membrane. Then, the membrane will be washed with radiolabeled DNA probes.
fluorescent or radioactively labeled tool that allows scientists to identify a specific sequence within a large sample. DNA probes are single stranded DNA, so they only hybridize with complementary DNA sequences.
essentially the same process as southern blotting. The main difference is that
northern blotting identifies fragments of a known RNA sequences using RNA probes.
similar process to southern and northern blotting. It is used to quantify amount of target protein in a sample. This is achieved by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE).
In SDS PAGE, proteins are denatured and given a negative charge by sodium dodecyl sulfate. The negative charge is proportional to the protein's mass. The actions of sodium dodecyl sulfate allow for better electrophoresis through the polyacrylamide gel - i.e. the gel that holds the proteins.
After SDS PAGE, the denatured proteins are transferred to a membrane just like in southern and northern blotting. However, these proteins are not treated with a nucleic acid probe. Rather, they are treated with primary and secondary antibodies.
The primary antibody has a target protein antigen. Therefore, the primary antibody will selectively bind to the target protein. The secondary antibody is attached to an indicator, which glows a certain color. It also has a primary antibody antigen. Therefore, it will selectively bind to the primary antibody.
Enzyme-Linked Immunosorbent Assay (ELISA)
technology to determine if a specific antigen exists in a person. It is beneficial because is aids in the diagnoses of certain diseases, like HIV.
The ELISA protocol is accomplished by taking blood from a person. Then, the antibodies from that blood are placed into a microtiter plate. If any of these antibodies bind to the antigen being tested for, there will be a color change in the microtiter plate.
The color change indicates the specific antigen must be present in the person, otherwise they would not have made the antibody for that antigen.
Adapted from: https://commons.wikimedia.org/w/index.php?curid=46451137
Pulse chase experiments
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).
study of all the genes present in an organism's genome. Specifically, genomics looks at the structure and function of genes. Genomics also examines how genes interact with each other in the genome.
way to store all the DNA of an organism's genome. This is achieved by using various types of restriction enzymes to cut the genome into many fragments. The fragments can then be cloned using PCR. DNA ligase inserts the cloned fragments into plasmids by catalyzing phosphodiester bonds between their ends.
The plasmid preserves the fragments from being broken down. It also allows for the plasmids to be screened for, using the antibiotic resistance or color-change methods. Once the appropriate plasmids have been isolated from the library, they can be cloned via bacterial cloning.
A DNA microarray
chip containing thousands of DNA probes that are complementary to a certain gene sequence. In this way, a cell's active transcription can be washed over the DNA microarray, and the DNA probes will hybridize with any gene they are complementary to. Fluorescence is emitted to let researchers know hybridization has successfully occurred.
This is useful because it allows researchers to determine which genes are expressed and which genes are not expressed in a type of cell. For example, a DNA microarray allows cancer researchers to see which genes are over/under expressed in comparison to a normal cell.
Typical microarray protocol:
1. Isolate a specific type of cell from a sample. Remove all the mRNA, because it represents the active transcription of that cell type.
2. Using reverse transcriptase, synthesize cDNA from the mRNA.
3. Hybridize the cDNA with the DNA probes on the microarray.
4. Use an analysis machine to examine the microarray for fluorescence.
models that researchers use to identify the function of a gene. Essentially, the idea is that a gene sequence will be taken from one type of organism, then it will be inserted into a different organism through recombinant DNA technology. This allows researchers to study the functional purpose of gene sequences.
Creating transgenic animals for the production of medications is a labor intensive process. For this reason, scientists have looked for ways to clone transgenic animals, such that they do not have to go through the process of creating and raising many different ones.
process of taking a somatic cell from an animal and producing a genetic copy from that cell. It is different from fertilization in that there is no genetic variation; each clone is a genetic replica of the parent animal.
different levels of gene expression on the tissue they are a part of; however, they all contain the full genome. The somatic cells involved with reproductive cloning are usually multipotent
● This describes a single cell with the ability to divide and produce an entire organism. Can produce extraembryonic membranes.
● A zygote, up to a morula, consists of totipotent cells. Any one of these cells can produce an entire organism.
A stem cell that can differentiate into any of the three germ layers: endoderm, mesoderm, or ectoderm.
● They can give rise to any cell type, but they cannot develop an entire organism because they can't develop extraembryonic tissue, like the placenta.
Dolly the sheep
first and most famous example of reproductive cloning. She showed that multipotent somatic cells could be reverted to totipotency. Further, she illustrated that somatic cells do contain the entire genome.
Dolly was made by taking the nucleus of a mammary cell, which came from the udder of an adult sheep. This nucleus was then inserted into another sheep's enucleated ovum, a totipotent cell that can give rise to an entire animal. In this case, the animal was Dolly.
There are three components of a chromatography apparatus:
1. The sample
2. The mobile phase
3. The stationary phase
sample dissolved in a solvent, and the solvent is considered the mobile phase (i.e. it can move). The mobile phase (containing the dissolved sample) will be placed in an apparatus that contains the stationary phase, which does not move (i.e. it is stationary).
Fluorescence Return After Photobleaching (FRAP)
researchers to see how and where biomolecules are moving in a live cell. T
Typical FRAP protocol:
1. A scientist measures the baseline fluorescence of a sample.
2. Then, an area of the sample is photo-bleached. Photo-bleaching causes pigmented
molecules to irreversibly lose their fluorescence.
3. Due to cellular dynamics and the moving cytoplasm within the cell, the photo-bleached
molecules are replaced by unbleached molecules over time.
4. This gradually restores fluorescence to the area.
Fluorescence Lifetime Imaging Microscopy (FLIM)
provides a quantitative measure of the concentration of various ions, molecules, and gases in a cell. This is achieved by irradiating cell samples with light and measuring their fluorescent lifetime.
YOU MIGHT ALSO LIKE...
Microscopy and Lab Techniques
Bio Lab Midterm
BIO 315 Exam 1 Ch. 18
Chapter 20: Biotechnology
OTHER SETS BY THIS CREATOR
DAT Chapter 15
DAT chapter 14