When is a plant cell plasmolyzed?
What is a hypertonic solution?
A hypertonic solution is a solution that has a higher concentration of solutes than found inside the cell
What are some of the functions of cell membranes?
Separation of cell contents from external environment
Organization of chemicals and reactions into specific organelles within the cell
Regulation of the transport of certain molecules into and out of the cell and its organelles
Beet cells red pigment, located in the cell's large central vacuole and surrounded by the tonoplast membrane
Molecule or part of a molecule that absorbs radiant energy (light)
Graph that shows the amount of light absorbed at a number of wavelengths
Four parts of a spectrophotometer
Device that isolates a photoelectric tube
Standard curve limitations
Standard curve is specific to the pigment and its buffer
Standard curve cannot be used for absorbances beyond the range of the standard curve
Cu = Cd/D
Deposits single bacterial cells from a liquid culture over the surface of an agar medium
Prevents bacteria in the environment from contaminating work, and prevents bacteria in work from contaminating the environment
Aspetic techniques that can be used
Sterilize surfaces and working surfaces
Washing hand before and after
Flaming inoculating loop and lip of culture tubes
Reduce time sterile medium, cultures, or bacteria are exposed to air
Work in area with low resident population of bacteria
What does the blank used with a spectrophotometer consist of?
How is a spectrophotometer zeroed?
Define each term in the "fluid mosaic model"
Fluid: membranes are able to move, things may pass through them (selective permeability)
Mosaic: membranes are composed of a variety of different things - phospholipid bilayer, enzymes, proteins that act as channels
Explain phospholipid bilayers
Fatty acid bilayers, where the hydrophobic ends of the fatty acids attract each other to the inside of the layer and the hydrophilic ends are on the outside of the membrane in the water, creating a double layer of fatty acids
How are bacterial species identified?
Cell and colony morphology, chemical composition of cell walls, biochemical activities, and nutritional requirements
What is the best way to isolate individual cells?
Streaking them onto an agar plate, so that each individual cell will produce a colony
Cell wall is composed of a thick layer of peptidoglycan surrounding the cell membrane
Differential stain to divide bacteria into Gram-positive and Gram-negatie
Steps of a gram stain
A basic dye (crystal violet) is used to stain the peptidoglycan in both cells, then iodine is used to increase affinity of the dye to peptidoglycan. Ethanol is then used to dissolve lipids in outer membrane of Gram-negative bacteria, allowing iodine dye complex to leave cells (peptidoglycan layer too thin to retain dye), while Gram-positive cells retain the dyed due to the thick layer of peptidoglycan - a counterstain is then applied that dies the Gram-negative cells pink
Commonly reocognized cell morphologies
Cocci: spherical shape
Bacilli: shaped like rods or cylinders (long and slender, or so short they resemble cocci)
Spirilla: resemble a corkscrew
What are three other ways to identify bacteria besides morphology?
Presence of flagella (motility)
Formation of endospores
How is motility in bacteria tested?
Bacteria are injected into a tube containing a dye that turns red when oxidized by growing bacteria- distribution of red dye indicates swimming ability
How is formation of endospores determined in bacteria?
Sample can withstand extreme conditions (high temperatures) and will grow at optimal conditions
What are the different enzymatic activities in bacteria that can be tested?
How can resolution be decreased (improved)?
Using an illumination source with a smaller wavelength
Increasing the numerical aperture of the objective lens, as well as using immersion oil with the 100x objective lens
How is the resolution value calculated?
Why do Gram-positive bacteria stain purple?
They contain a thick layer of peptidoglycan which retains the iodine-crystal violet complex CV-I, causing cells to hold the dye and thus retain the purple colour even after the ethanol wash
Why do Gram-negative bacteria stain pink?
Gram negative cells have only a thin layer of peptidoglycan, so the CV-I complex easily washes out, and thus the cells are able to be counterstained pink because they no longer contain any crystal violet dye
What is the effect of high temperatures on bacteria that do not form endospores?
High temperatures will kill cells that do not produce endospores as it can damage cell membranes and denature proteins, resulting in cells that are unable to function
Microtubules, microfilaments, and intermediate filaments which function in cell structure, cell motility (flagella and cilia - microtubules as part of their ultrastructure), and various biological processes
Mitochondria, chloroplasts, Golgi apparatus, endoplasmic reticulum, nucleus, and vacuoles/vesicles
Function in cell motility (flagella and cilia) and one organism also uses cilia to propel food towards its oral groove
Used in amoeboid movement and organelle movement (intermediate filaments give nucleus its shape - nuclear lamina)
How do microfilaments act in amoeboid movement?
Phase contrast microscope
Phase contrast microscope
Cells and medium have different refractive index, and therefore light traveling through a material with different refractive indexes show a change in phase of light waves, which the microscope then translates to a change in light intensity (areas of higher refractive index appear darker)
Some cell structures contain autofluoresce, some require staining - by exposing cells to several stains at once, different structures will fluoresce different colours
How can contrast be improved?
Staining compounds (vital stain - living cells/tissue, or dead cells and tissues), using special types of microscopes to manipulate light, and by reducing the amount of light
What effect would cytochalasin (inhibits microtubules) and cholchicine (inhibits microfilaments) have on Pelomyxa?
Cytochalasin would cause amoeba to become sessile, as the microfilaments are responsible for the movement of amoebas - cholchicine would have no effect
What effect would cytochalasin (inhibits microtubules) and cholchicine (inhibits microfilaments) have on Euglena?
What effect would cytochalasin (inhibits microtubules) and cholchicine (inhibits microfilaments) have on motile prokaryotes?
What is phagocytosis, and how does it differ from receptor-mediated endocytosis?
Phagocytosis is the process that Paramecium use to take in food. It differs from receptor-mediated endocytosis in that receptor-mediated endocytosis is very specific and allows the cell to acquire bulk quantities of specific substances, whereas phagocytosis is more general and can take in different substances
Organelles (enzymes) that digest or break down waste materials and cellular debris, such as worn out organelles, food particles, and engulfed bacteria and viruses
Contains cell's genetic information, and is surrounded by the nuclear lamina (made up of intermediate filaments) to protect the DNA
Engulfs food via phagocytosis and uses lysosomes to break the food down
Organelles found in plant cells that are used in photosynthesis - they capture light and convert it to usable energy
Proteins that catalyze metabolic reactions without being consumed or destroyed by the molecule - lower a reaction's activation energy (substrate specific)
Molecule to be reacted, that fits into a uniquely shaped pocket of the enzyme called the active site and binds with the enzyme as it is converted into the end product
Allows plants to use starch it has stored after photosynthesis - takes amylose and breaks it down into smaller molecules by hydrolysis (glucose molecules, maltose, and shorter chains of amylose)
Polymeric macromolecule composed of glucose monomers that is too large to pass through a cell membrane
What are enzymes made up of?
Enzymes are proteins, which are made up of amino acids
What is an active site?
An active site is a site that uniquely fits the substrate specific to the enzyme, and will activate the enzyme once the substrate binds to the site
Energy transfer from light to chemical bonds through series of light reactions
What series of reactions occurs in photosynthesis?
Light energy from sun strikes pigments in thylakoid membrane of chloroplast, which is transformed into excited electrons (electrical energy), then into chemical energy in the form of bonds in ATP and NADPH molecules, and the ATP and NADH molecules are then used to power the fixation of carbon dioxide into sugar molecules (Calvin cycle, occurring in stroma)
Determines which wavelengths of light the chloroplasts maximally absorb (these wavelengths also produce the highest rates of photosynthetic activity)
Chloroplast suspension is mixed with indicator dye DCPIP - as DCPIP accepts electrons from the electron transport chain of Photosystem II it becomes reduced and therefore colourless, allowing absorptions to be measured to determine concentrations
What were the controls in the photosynthesis in spinach chloroplasts experiment?
Controls must show that DCPIP is stable and there is no other source of electrons to reduce DCPIP (colour does not change spontaneously) and that the colour of chloroplast suspension is stable and does not change colour spontaneously
What are the independent and dependent variables for the absorbance spectrum of photosynthesis in spinach chloroplasts?
The independent variable is the wavelength of light, and the dependent variable is the absorbance of the chloroplast suspension at varying wavelengths of light
What are the independent and dependent variables for the action spectrum of photosynthesis in spinach chloroplasts?
The independent variable is the colour of light, and the dependent variable is the absorbance of the solution
Why are spinach leaves green?
Spinach leaves are green because they maximally absorb blue and red wavelengths, and green wavelengths are absorbed the least and are therefore reflected back the most, resulting in the green colour
At what wavelength is the action spectrum measured at?
The wavelength that photosynthesis occurs at maximally
Where do light reactions take place in the chloroplast? Reactions of the Calvin cycle?
Light reactions take place in the stroma of the chloroplast, and reactions of the Calvin cycle take place in the stroma
Measuring oxygen levels, sugar produced, or carbon dioxide levels
How is ATP produced?
Through the catabolism of carbohydrates, proteins, and fats
Glycolysis in eukaryotes
Cytosolic reactions to convert glucose to pyruvate (one molecule of glucose results in the net production of 2 molecules of ATP via substrate level phosphorylation
After glycolysis, what occurs in the presence of oxygen?
Eukaryotes convert pyruvate into acetyl CoA, which is transported to Kreb's cycle in mitochondria (produces 2 more molecules of ATP) and then oxidative phosphorylation (the transfer of electrons from food to oxygen) produces the rest of the ATP molecules (carbon dioxide is also formed as a by-product)
After glycolysis, what occurs in the absence of oxygen?
Pyruvate is degraded via a series of cytostolic pathways - lactic acid fermentation and alcohol fermentation (produces ethanol and carbon dioxide, regenerates NAD+ - required for glycolytic pathway)
What sort of feedback system occurs in alcohol fermentation?
Fermentation of glucose produces ethanol, but high concentrations of ethanol are toxic to yeast
Physiological response curve
Why is fermentation necessary?
Why was the yeast flask swirled prior to adding yeast to each tube?
To re-suspend the yeast and therefore the ensure that similar concentrations of yeast were present in each tube (constant)
What would happen if the metabolism in yeast experiment were done without the 10 minute pre-incubation period?
The lag phase of the physiological response curve would be significantly longer as the pre-incubation period brings the tube to a temperature at which yeast metabolizes glucose most effectively, and therefore without the incubation period the yeast would not metabolize glucose as well
What process are the yeast in the Durham tube undergoing?
The yeast are undergoing fermentation - other eukaryotes undergo aerobic respiration, and only prokaryotes undergo anaerobic respiration
What metabolic processes occur in the cytoplasm?
Alcohol fermentation, ATP production, glycolysis, and NADH production
What metabolic processes occur in the mitochondria?
ATP production, Krebs cycle, electron transport chain, and NADH production
Bacterial genomic DNA
Consists of a double stranded DNA helix arranged in a circle that is anchored to the bacterial plasma membrane - 4000 genes that encode all the functions of the bacterial cell
Bacterial plasmid DNA
Floats freely in cytoplasm of bacterial cell
Circular and can assume supercoiled conformation in which circular double helix molecule twists on itself
Much smaller than genomic DNA (2- 25 genes)
Can sometimes conform extra properties to the cell that allow the cell to survive in conditions that it could not survive without the plasmid DNA (only when there is selective pressure)
Arranged in linear strands (chromosomes - 23 pairs) in nucleus of cell (30 000 - 35 000 genes - high molecular weight DNA)
Can be used to analyze small amount of plasmid DNA - DNA is not very pure and maxi prep must be used for further analysis as it is a larger quantity of very pure DNA - separates plasmid DNA from bacterial genomic DNA based on size and conformation
How can high molecular weight (HMW) DNA be extracted?
High affinity for glass - buffer solution must contain Tris and EDTA as it binds magnesium ions which are required for DNAse, preventing DNAse from functioning and degrading the DNA into nucleotides
What does centrifuging do?
Creates a centrifugal force that causes bacterial cells to collect in a pellet at the bottom of the tube - liquid above is referred to as the supernatant
What does vortexing do?
Vortexing disrupts the pellet of cells so that they may be re-suspended
Why is STE added to the DNA treatment?
Washes the medium away from the cells
What is Solution I in the DNA treatment?
A buffered, isotonic solution that is used to re-suspend bacterial cells
What is Solution II in the DNA treatment?
Contains sodium dodecylsulfate (SDS) and sodium hydroxide (an alkali) - SDS denatures proteins and disrupts the plasma membrane, causing the cell to lyse and releasing cell components into the solution, and NaOH raises the pH of the lysate to denature the hydrogen bonds between the base pairs of DNA, separating the helix
What is Solution III in the DNA treatment?
Acidic potassium acetate solution that neutralizes the pH in the lysate so that some hydrogen bonds in the DNA will re-form in random base pairs, resulting in a tangled, insoluble mass of DNA - hydrogen bonds in the plasmid DNA reform between the original complementary base pairs (when solution is placed on ice potassium forms white, insoluble mass with SDS that precipitates out along with many of the proteins, cell wall, debris, and genomic DNA)
What does centrifuging do to the genomic DNA-potassium-SDS-protein-cell wall complex?
Causes the complex to pellet in the bottom of the tube and the plasmid to remain in the supernatant solution
What does the 95% ethanol wash do in the DNA treatment?
Removes water molecules from macromolecules by decreasing hydrogen bonding between water molecules and macromolecules (plasmid DNA and RNA come out of solution and precipitate, so that they may be centrifuged into a pellet)
What does the 70% ethanol wash do in the DNA treatment?
Removes the salts which were not removed with the 95% ethanol, and hydrates the pellets slightly so that it may dissolve in the aqueous solution
Why is the 30 minute incubation period necessary in the DNA extraction?
Why is sodium acetate used to precipitate HMW DNA?
The salt ions compete with macromolecules (DNA) for the water molecules
What are the 2 properties of DNA that allow you to separate genomic DNA from plasmid DNA
Size - big genomic DNA precipitates faster with centrifugation
Conformation (shape of molecule) - supercoiled plasmid DNA maintains its shape even when hydrogen bonds in backbone are broken
How can HMW DNA be extracted from solution?
Its affinity for glass and the fact that it forms very long "threads" of DNA
What would happen if the tube were vortexed after the addition of Solution II?
Genomic DNA would break and would not all be centrifuged out, therefore contaminating plasmid DNA
What is the difference between genomic DNA, plasmid DNA, and eukaryotic DNA?
Genomic DNA contains the majority of genes needed for the bacterial cell to function
Plasmid DNA is a small, circular structure of DNA in the cell cytoplasm that contains genes that can allow the bacteria to survive in conditions where it could otherwise not survive
Eukaryotic DNA is much larger and is contained within the nucleus, in 23 pairs of chromosomes, encoding all the genes necessary for the survival of the eukaryote
What is the purpose of the 95% ethanol and 70% ethanol wash?
95% ethanol dehydrates the cell
70% ethanol treatment removes the salts and rehydrates the plasmid DNA, allowing it to dissolve faster
Why must plasmid DNA be kept on ice following incubation?
DNAse will break down DNA at room temperature - T solution has EDTA to inactivate DNAse
What was the experiment performed by Avery, MacLeod, and McCarty?
Tested various cellular macromolecules for their ability to transform non-virulent Streptococcus pneumoniae into virulent bacteria - discovered DNA was the only macromolecule capable of transforming non-virulent bacteria into virulent bacteria
How did the experiment performed in Biol 107 differ from Avery et al?
Escherichia coli was used
E. coli was examined for transformation by a gene on the plasmid DNA instead of the genomic DNA
E. coli cells needed to be made competent to uptake DNA using a calcium chloride solution
Mice were not used (medium containing kanamycin was used instead)
Only DNA was focused on (as opposed to various parts of the cell)
How were kanamycin sensitive E. coli cells made proficient to take up DNA?
A calcium chloride solution was made, which created holes in the cellular membranes (competent cells)
How is a competent cell transformed?
If plasmid DNA entering competent cells is capable of replicating, the competent cells will be genetically altered or transformed (kanamycin resistant) - all descendants of transformed cells should be genetically altered
What is kanamycin?
Antibiotic belonging to the family of antibiotics characterized by their ability to inhibit protein synthesis in prokaryotic cells - they are transported into the cell by oxygen dependent active transport system and irreversibly inhibit protein synthesis by binding to a small subunit of ribosomes in bacterial cell, so cells are unable to synthesize proteins - cell death
What occurs in kanamycin resistant cells?
Phosphotransferase enzyme is encoded and expressed in the presence of kanamycin, which phosphorylates (adds a phosphorous group) to kanamycin and renders the antibiotic inactve
Use of a DNA template to synthesize RNA
Reading of mRNA to produce protein
Plate Count Method
Viable cell count (living cells only) in which original cell suspension is diluted into suspensions of decreasing cell concentration, which are spread onto the surface of an agar medium and allowed to incubate so that single cells may grow into a colony - following incubation colonies may be counted, and each is representative of a single cell originally deposited on the plate
Petroff-Hausser Counting Chamber
Total cell count (living and dead) using a specially designed microscope slide with a depressed surface and etched grid, where a thin layer of cell suspension of known volume is spread and the number of cells in the volume is directly counted with the aid of a microscope
Optical Density (OD)
Indirect method of total cell count, measuring turbidity (cloudiness of a solution due to the presence of particles such as cells), measured using a spectrophotometer, and developing a standard curve
Why is Solution T (Tris) buffer used in the Transformation of Bacterial Cells lab?
Maintains the pH at 8.0 and is the solvent for plasmid DNA
Solution B is the solution used to dissolve DNAse but does not contain DNAse
What occurs during the first incubation period of the Transfomation of Bacterial Cells lab?
What occurs during the heat shock incubation period in the Transformation of Bacterial Cells lab?
Helps the plasmid DNA enter the competent cells and induces the expression of survival genes necessary to repair damage to the plasma membrane
What does the third incubation period in the Transformation of Bacterial Cells lab do?
Allows time for kanamycin resistance gene to be expressed - must be transcribed into mRNA, then mRNA must translate it into a polypeptide chain (phosphotransferase)
The plate which does not contain plasmid DNA, and instead contains solution B, Tris buffer, and competent cells
The plate that contains plasmid DNA that has been broken down into nucleotides by DNAse, as well as solution B and competent cells
There would be colony growth on plate 5+K, as the DNAse cannot enter the competent cells and the plasmid DNA would not have been broken down - the kanamycin resistant gene would have been expressed in the competent cells
What would occur if the environment in which the E. coli was grown was anaerobic?
The kanamycin would not affect the growth of the cells, as kanamycin enters the cell in an oxygen dependent manner