Lab Exam 1 Flashcards
Covers Labs 1 thru 4.
Substances Used in Labs 1 thru 3 (4)
Methocel: Paramecium in Lab 2; slows down protists by making medium more viscous
FCCP: Nitella in Lab 2; slows down cytoplasmic streaming by removing the proton gradient in chloroplasts, thereby stopping ATP production
Amylase: Lab 3; found in saliva; breaks bonds of glucose (from starch) into maltose
DNS: Lab 3; pH 14, immediately denatures the amylase; allows for indirect measuring of maltose created via color change at high temperatures
Substances used in Lab 4 (6)
DCMU: Lab 4; uncharged and hydrophobic; blocks proton acceptor (specifically plastoquinone), thereby interrupting ETC and inhibiting photosynthesis; DCPIP should not change
DCPIP: Lab 4; blue; acts as artificial electron acceptor, thus allowing the ETC and photosynthesis to resume —turns into DCPIPH2 (colorless) during photosynthesis → therefore, can determine that photosynthesis is happening as the solution goes from blue to clear
Methylamine: spinach in Lab 4; can enter thylakoid lumen because it is an uncharged weak base; removes proton gradient in chloroplasts, thereby stopping ATP production
Sodium phosphate buffer: Lab 4; no sucrose; ruptures envelope membranes of chloroplast due to lack of osmoticum BUT thylakoid membranes remain intact
Sucrose phosphate buffer: Lab 4; maintains constant pH, acting as an osmoticum that prevents chloroplasts from rupturing
Acetone: Lab 4; hydrophobic; used to dissolve pigments
Describe the relationship between pKa and pH.
High pH = super basic, lacking of H+ ions floating around; most likely going to be protonated
Low pH = super basic; lots of H+ ions floating around; most likely going to be deprotonated
If substance has higher pH than surrounding, substance will be protonated; if not, will be deprotonated.
–
pH = pka - log( [A] / [HA] )
If pKa > pH, then deprotonated
If pKa < pH, then protonated
Define the following:
- Reduction
- Oxidation
- Blank
- Control
Reduction: gain of electrons in the form of H+
Oxidation: loss of electrons in the form of H+
Blank: baseline absorbance reading of your reagent solution
Control: assures the experiment is working properly (ie. knowing the standardized concentration)
What is the role of buffers and when do we add them?
Buffers keeps the pH neutral (~7), which is the level where biochemical processes happen; they are added BEFORE the enzymes in order to keep the pH at a functional level (as enzymes will mess up the pH of the system otherwise).
Micropipette
- How to Use
- Errors
Proper usage: first stop to extract; second stop to deploy
If second stop used to extract, a larger range of error is expected (therefore, more variability in the readings).
Acceptable error range is 3%
Spectrophotometer
As the concentration of the solute increases, transmittance decreases and absorbance increases.
Beer’s Law: Abs = clz = -log (T) where T = I / Io
Three important parameters in microscopy
Magnification: increasing sizes by changing lenses
Resolution: clarity and sharpness (aka the ability to “see” two different objects) via lens design
Contrast: juxtaposition of unlike elements to achieve a strikingly differentiation by changing lighting
Low power magnification has LARGE depth of focus. → As you increase the magnification, the depth of focus gets SMALLER, field of view gets SMALLER, amount of light DECREASES. (Math: increase magnification by tenfold, decrease depth of focus by tenfold as well.)
Important and Obscure Parts of the Microscope to know + Functions (8)
Field diaphragm: controls the amount of light that reaches a specimen
Aperture diaphragm: regulates how much light passes through; can improve contrast by closing
Condenser: focuses the light on the specimen
Eyepieces: magnification of 10x — Right: focus first using coarse; contains the reticle (measuring device consisting of 100 subdivisions) ++ Left: focus second using diopter; contains pointer
Diopter: used to compensate for the focusing differences between your two eyes
Resolving power: ability to distinguish between two objects
Numerical aperture (of microscope objectives): largely determines the resolution; higher NA = better resolution
IN A FLUORESCENT MICROSCOPE → Dichroic mirror: reflects at a certain wavelength and transmits at a longer wavelength
Types of Dyes (2)
Fluorescence: very specific; helps with outlining pathways
Brightfield: Lab 2 on cheek cells; general; helps to see entire cells by staining with dyes for contrast; requires cells to be fixed / preserved (aka kills them)
What to include in your specimen drawings
- Drawing of specimen
- Labelling of anything you can positively / confidently identity
- Name and total magnification of specimen
- Scale
Common characteristics of living organisms
- all organisms are made up of a single cell or many cells
- cells have precise, programmed molecular mechanisms for reproduction and metabolism
- different, specialized cells often have different amounts of these organelles to accomplish specialized functions
Define protist.
heterogenous
need sand on slide to prevent squashing; have contractile vacuole (stores and excretes waste and excess water by merging with the cell membrane to dump its contents outside the amoeba)
eg. amoeba, paramecium, volvox
Identify the following:
- Human cheek cells
- Nitella
- Elodea
Human cheek cells: stained with methylene blue (brightfield); nucleus stands out
Nitella: cytoplasmic streaming via microfilaments; treated with FCCP to slow down streaming
Elodea: cytoplasmic streaming; slower than Nitella
Identify the following:
- Amoeba
- Paramecium
- Volvox
ALL ARE PROTISTS.
Amoeba: pseudopodia via microfilaments; assists with cytoplasmic streaming
Paramecium: cilia; stained red and treated with methocel to slow it down
- Oral groove: area where cilia sweeps food and nutrients into; when filled enough, breaks away to form food vacuole inside cytoplasm
- Micro/macronucleus: contain full complement of genes with hereditary information → cannot survive without macro, cannot reproduce without micro
Volvox: flagella; circular; photosynthetic via chloroplasts
Cytoplasmic streaming (2)
+ refers to the growing part, - refers to the shrinking part → NOT CHARGES
Microfilaments: Actin (+/ - ends) versus myosin (motor protein)
- Primarily for pseudopodia (as in amoeba) and cytoplasmic streaming in Nitella
Microtubules: tubulin dimers (+ / - ends) versus denien ( - motor) and kinesin (+ motor)
- originate from the Microtubule Organizing Center (MTOC)
- Primarily for cilia and flagella
Epithelial Cells (5)
separate the inside of an organ from the outside; mostly dead already
- Simple = one layer of cells
- Stratified = multiple layer of cells
- Squamous = wide in epithelium, thin perpendicular to it
- Columnar = thin in epithelium, elongated perpendicular to it
- Cuboidal = same size in both planes/dimensions
Surface Area to Volume Ratio
Must be optimized and very large.
Volume increases by cubed dimension but surface area is only squared
Volume is consumption and surface area is production .
Define bioluminescence.
in bacteria (specifically vibrio fischeri) – arises out of the release of n acyl homoserine lactose, which occurs when the concentration of this reaches a critical threshold inside the bacteria
Streaking guidlines
Procedure: sterile loops for each streak; make sure you go back onto the old streak → goal to isolate the bacteria by spreading them out; done twice
- Rapid growth over time but nutrients can be depleted and waste products can be formed.
- Colony should be genetically identical is only one bacteria founded the colony
Types of Medium (4)
support growth, provide osmotic balance, and acts as a pH buffer to bacteria → eg. SWC (sea water complex) is a complex, selective medium
- Defined: all components known and can be manipulated
- Complex: components are known but specifics unknown or slightly vague
- Selective: contains ingredients that favor one species over another
- Differential: can diagnose infection or show phenotypes; not used or discussed in lab
Protein Structures (4)
made of amino acids polymerized together to form peptide bonds
- Primary: linear and planar connectivity
- Secondary: involves backbone C; has the potential to create H bonds → do NOT involve the R
- - Beta pleated sheets: can be parallel or antiparallel
- - Alpha helix: - Tertiary: involves the R group
- Quaternary: interaction between polypeptides
Lab 3B, Part 1: Adjusting Enzyme Concentration
- Graph descriptions
- Safeway Example + 3 portions
OD versus enzyme concentration: linear
OD (enzyme) versus enzyme: horizontal; same as linear when converted
Safeway example
– Saturation: all active sites working at maximum capacity, therefore creating a plateau (aka Vmax)
- First linear portion: enzyme reaction proceeds at a constant rate as long as the substrate is in excess, where the slope of the line is proportional to the enzyme concentration
- Second non-linear portion: reduced reaction rate as concentration of substrate decreases and becomes limiting as the reaction continues to consume it
- Third flat portion: aka plateau; all active sites are at maximum capacity and increasing the substrate concentration has no effect
Lab 3B, Part 2A: Varying Temperature
Enzymes are changing (specifically, lowering) the energy of activation
Lower temperatures do not provide enough energy because the molecules are moving too slowly to react.
Higher temperatures can initiate denaturation (change or breaking away from the optimum structure) of enzymes OR moves the particles around too much for proper reaction
** Temperature chart is a bell curve because optimum is in the middle.
Lab 3B, Part 2B: Varying pH
Throwback to unprotonated / protonated explanation in lab
Most enzymes and biochemical reactions work best at a pH of 7 (bell curve again)
R groups can change with the pH which is BAD because now the experiment is not operating under the best / most stable conditions
Extreme pH values can result in enzyme denaturation, but smaller changes may affect the reaction rate
Define the Michaelis constant.
Km: concentration at which Vmax is halved; does not increase with enzyme concentration
Anatomy of a Leaf (4)
Upper epidermis: secretes waxy cuticle to prevent water loss and osmotic exchange
Mesophyll: photosynthetic cells; contains chloroplasts
- Palisade: near upper; cuboidal and neatly packed; no room for gas exchange; absorb radiant energy via chlorophylls
- Spongy: near lower; round with lots of room; allows for gas exchange with CO2
Vein: carries water and other soil-based nutrients
Lower epidermis: also has waxy cuticle BUT allows osmotic exchange thru presence of stomata and guard cells (which open and close in response to osmotic pressure)
Anatomy of a Chloroplast (4)
Outer membrane: phospholipid bilayer
Inner membrane: phospholipid bilayer
Stroma: space between inner membrane and the thylakoid lumen
Thylakoid: phospholipid bilayer as membrane; contains the chlorophyll
- Granum: stack of thylakoids
- Lumen: inside of thylakoid membrane
Photosynthesis
- Photosystem 1
- Photosystem 2
- ETC
- Proton Gradient
- Creating NADPH
- Notes
Let’s start with PHOTOSYSTEM I:
- Light passes through in the form of radiant energy / photons which are absorbed by pigments (chlorophyll A/B + carotenoids, which all function at different wavelengths) inside the antenna complex.
- - Pigment molecules located close to one another so that light energy is transferred from one to the other → Resonance energy transfer: give to a neighboring chlorophyll molecule - BUT the energy they pass on is always less (shorter wavelengths) because of the decay by successive electron transfers that emit energy and longer wavelengths (closer to red)
- Eventually, this energy is brought to a special set of chlorophyll A in the reaction center which can absorb P700 wavelengths (red)
Filling the hole with PHOTOSYSTEM II:
- Hole created when P700 depletes itself of electrons and new ones are provided by Photosystem II.
- Similar procedure as in PS I but instead uses specialized chlorophyll a molecule of P680, which has an electron acceptor that initiates the ETC from PS I to PS II
- - Creates a high energy electron
Explaining the ELECTRON TRANSFER CHAIN:
- Each high energy electron goes through the ETC. ETC includes the hydrophobic organic compound plastoquinone (Pq) and cytochrome protein complex plastocyanin (Pc).
- - Both of these are reduced and then oxidized as the electrons pass along this chain.
- - Both use the energy from these electrons to transport H+ from the chloroplast stroma to the thylakoid lumen (where [H+] increases and thus, pH decreases) via osmosis.
Creating the PROTON GRADIENT:
- Water is brought via veins.
- Cascade of ETC (releases energy)+ enzyme + water = split water into oxygen, hydrogen ions, and electrons
- - Oxygen considered waste product and goes back into atmosphere.
- - Electrons (high energy) replenish hole created in PS II.
- - Hydrogen ions are released into the lumen. - The energy provided by the H+ concentration gradient powers the production of ATP (phosphorylation of ADP by ATP synthase in the thylakoid membrane).
- - Excess hydrogen ions are formed by 2 sources: the energy used in ETC by Pq and Pc, and the splitting of water
Alternatively, creating NADPH:
- Light energy has been used to boost energy of / excite electron in P700 chlorophyll a dimer. This electron is transferred to electron acceptor in reaction center of PSI.
- Electrons passed to second ETC (including ferredoxin (Fd)).
- Fd passes two e- to NADP+ to form NADPH + H+. This is used in the Calvin Cycle to form sugar. The 2H+ are located in the stroma.
Note:
- Exciting an electron to high energy in PS I allows for the creation of NADPH by passing to Fd.
- This is different from exciting an electron to high energy in PS II, which allows for the creation of lots of H+ ions by splitting water and using Pq and Pc to bring in H+ ions from the stroma.
- Electron transport and H+ transport are coupled in the ETC (ie. H+ from the stroma into the lumen)
Give two points about fluorescence.
Every reaction releases heat and lengthens the wavelength until the length matches the red one (which is the longest wavelength).
Excited electrons can donate electrons BUT if there is no neighbor to pass it onto, so energy is released in the form of fluorescence (therefore, heat and longer wavelength).
Explain the use of chromatography in Lab 4.
- Chlorophyll A has a methyl group
- Chlorophyll B has an aldehyde, therefore more hydrophilic (therefore more polar)
THEREFORE: on chromatography paper, chlorophyll B is lower than A; both however, are lower than the carotenoids (yellow)
** Chromatography paper moves non polar (hydrophobic) more than polar (hydrophilic)
Identify three specific lab procedures you should know from Lab 4.
Blender: allows rupturing of cell walls / membranes BUT coupled with sucrose phosphate buffer allows for maintaining of chloroplasts
Centrifugation: separates substances into pellet (mostly chloroplasts / denatured proteins) and supernatant (organelles, buffer, etc – basically the hydrophobic photosynthetic pigments)
Standardization: dilutes original “enriched” suspension to a level where reaction rates can be measured; also allows for comparison between groups in the entire lab
– Steps from lab manual: Enriched + sucrose phosphate → brush to resuspend, ice to preserve photosynthetic activity → phosphate buffer (no sucrose) to rupture → measure absorbance, which is reflective of the number of chloroplasts → convert as needed to desired concentration
Hill Reactions (5)
- Reactions run
- Oxygen production
- ATP production
- Blank
- Dark – should’ve had no change; control to take into account background reduction
- Light: rate increases
Light + DCMU: rate increases a bit; OD drops just a little - Light + methylamine: rate increases a LOT; OD drops like crazy
- Methylamine: acts as a pH buffer and removes the H+ gradient
What about oxygen production? ATP?
- Blank = N/A
- Dark = none; none again
- Light = some; some
- Light + DCMU = none; none
- Light + methylamine = a lot; none
