Bio 107 Lab Lab 1
Bio 107 Lab Lab 1 – Introduction, Microscopes, Scientific Method and Osmosis Pure Culture – isolated group of genetically identical cells Aseptic Technique – sterile technique to reduce contamination of microorganisms into a pure culture, and to prevent bacteria from hurting you/environment, sterilize, reduce exposure to air time, work in clean areas Colony – microorganisms that have grown and divided into a small mass of genetically identical cells, large enough to be seen with naked eye Medium – solution/solid containing nutrients so that cells can grow and divide Swabbing Surfaces in Lab: all have microorganisms, potential contaminants Conversions: 1 mm = 1000 mm 1 mm = 1000 nm Light Microscope – light travels through light source, illuminates specimen and creates an image which is magnified by lenses Condenser Lens – focuses light from light source on the specimen, under the stage, has knobs that move it up and down Objective Lens – magnifies the image of the specimen, projects image into body tube, on rotating nosepiece, 4X, 10X, 40X and 100X Ocular Lens – magnifies image and inverts it for better viewing (10X in lab), aka eyepiece, contains ocular micrometer Light Source – lamp Condenser Diaphragm – controls amount of light entering lens system, reducing light improves contrast (lever under stage) Stage – horizontal, slide placed on it Coarse Adjustment Knob – moves stage up and down to bring into focus, with 4X and 10X only Fine Adjustment Knob – brings sharper focus, used at all powers Body Tube – hollow, light travels from objective to ocular lens, prism to direct light rays Total Magnification = MagnificationOcular Lens x MagnificationObjective Lens Resolution – capacity to distinguish two adjacent points as being distinct Resolution Value – minimum distance required between two points to remain identifiably separate Scientific Method – series of steps taken to answer a specific question of interest 1. Formulate an idea or question (usually comes from an observation) 2. Formulate a hypothesis – educated guess answer to your question based on prior knowledge 3. Test hypothesis by observation and/or experimentation. Variable – one things different between groups in a test Experimental Group – tests effect of independent on dependent variable Control Group – examines dependent without independent variable Independent Variable – manipulated variable of interest Dependent Variable – responding, measured result of manipulation Constants – factors kept the same between groups, independent variable should be only difference between experimental and control
Solvent Control Group – examines the solvent, tests that solvent doesn’t affect dependent variable 4. Draw a conclusion based on data obtained. Either refute or support your hypothesis (not proven). 5. Hypothesis to theory (after many test of a hypothesis) Osmosis – water movement across a semipermeable membrane, passive transport, water moves from an area of low solute to high solute concentration Cell survival depends on their ability to regulate influx and efflux of water Cell walls constrains size of cytoplasm, prevents membrane rupturing Hypotonic Solution – lower solute [ ] than cell cell swells – turgid Hypertonic Solution – higher solute [ ] than cell water leaves cell – plasmolysis Isotonic Solution – same [ ], no net mov’t of water reduced pressure – flaccid Marine algae should be able to handle higher [ ] salt solutions (Tetraselmis: motile, tiny, round) Fresh water algae should only be able to handle lower [ ] salt solutions before dying from plasmolysis (Mesotaenium: longer ovals) Lab 2 – Streaking Bacterial Cells, Membrane Structure and Function Membrane functions: separation from external environment, organization into organelles, and regulation of transport Role of structure studied by disrupting one or more of its functions Used temperatures to disrupt membrane function in Beta vulgaris Betacyanin – red pigment located in central vacuole Tonoplast – membrane surround central vacuole Plasma membrane – surrounded cell If these membranes damaged – betacyanin will leak out of cells and increase concentration of betacyanin in surrounding solution (measured indirectly and quantitatively by absorbance value) Spectrophotometry: Objects appear coloured because they absorb some of the visible spectrum and reflect the other wavelengths (cause their colour) Chromophore – part of a molecule that absorbs energy from light to excite its electrons, wavelength causing highest absorbance unique to chromophore (set spectrophotometer to specific wavelength of choromophore) Can create Absorption spectrums showing amount of light absorbed at different wavelengths Can measure concentration based on absorbance measurements Four parts: Light source is a tungsten filament lamp emitting white light, which is split into different colours by a prism, slit selects one wavelength of light, then the photoelectric tube compares incident light to transmitted light. Transmitted light:incident light = Transmittance (T), %age Absorbance (A) – negative log of transmittance, wavelength subscript Standard Curve – graph of absorbance and concentration, absorbancies measured with dilutions of a stock solution (known [ ]), specific to the pigment and buffer, cannot be
extrapolated (have to do a dilution of a solution with unknown concentration if its absorbance is beyond your standard curve) Dilution (D) = volume of original solution / volume of original + volume of solvent Cundiluted = Cdiluted / D Zero – set transmittance to zero with nothing in spectrophotometer Calibrate – set absorbance to zero with blank (all components of sample except molecule of interest) Optical Density – amount of light that is scattered or absorbed by particles suspended in solution, measures [ ] of cells in suspension Beet experiment: detailed in lab report Liquid Culture – suspension of millions of cells in a liquid medium with all nutrients required for growth Streaking – way to isolated single bacterial cells from liquid culture, 3 streaks each coming from each other, use aseptic technique (ex: flame inoculating loop), third streak will have isolated colonies of different types of bacteria Turbid – not clear, cloudy solution of for example, bacteria Lab 3 – Identification of Bacterial Species Identification of species based on: cell and colony morphology, chemical composition of cell walls, biochemical activities and nutritional requirements need pure culture by isolation (Streaking) GramPositive Bacteria – thick layer of peptidoglycan surrounding its one membrane, stain purple GramNegative Bacteria – thin layer of peptidoglycan, 2 phospholipid bilayers, outer membrane surrounding plasma/inner membrane, stain pink Gram stain – produces a colour result dependent of the cell wall structure of the bacteria, a differential stain dividing the bacteria into two groups 1. Stain – Crystal violet – picked up by cell wall of cells fixed to a slide, dyes peptidoglycan purple 2. Mordant – Iodine – increase the affinity of the dye to the peptidoglycan by forming large complexes with crystal violet, crystals in peptidoglycan 3. Decolorizing Agent – Ethanol – dissolves lipids in outer membrane of gram negative, wash off CVI complexes after 10 seconds, gramnegative turn colourless, the differential step (CVI will wash off grampositive but will take a longer time) 4. Counterstain – Safranin – pink/reddish dye that stains gramnegative, gram positive still purple GramPositive GramNegative Length of Exposure Crystal Violet Purple Purple 1 minute Gram’s Iodine Purple Purple 1 minute Ethanol Purple Clear 10 seconds Safranin Purple Pink 1 minute Colony Morphology – shape, size, edge, colour, surface texture, and elevation Cell Morphology – shape, arrangement, external structures, and gram stain Cocci – spherical
Bacilli – rod or cylinder shape, long and slender or short Spirilla – corkscrew shape arranged singly, in pairs, cluster, chains presence or absense of flagella, capsule, slime layer, fimbriae, pili Biochemical Test – many tests need to be performed to determine species of bacteria Motility Test – will swim away from area of injection to lower [ ] of bacteria using one or many flagella, the bacteria if they move will oxidize tetrazolium salt to red, if no movement will just be red along stab site Endospore Test – some grampositive and one gramnegative species can form endospores, endospores are highly resistant, dehydrated cells with thick walls and layers, can resist extreme temp., inoculate at 80 degrees and only endospores live, endospores is turbid, none is clear Glucose Fermentation Test – some bacteria metabolize glucose for their carbon energy source, medium with glucose and pH indicator phenol red will turn yellow if glucose is metabolized, if CO2 is made, bubbles will get trapped in inverted vial Tryptophanase Test – some bacteria metabolize the amino acid tryptophan for nitrogen, reaction is catalyzed by enzyme tryptophanase, end product reacts with DMCA and turns blue, if the cells don’t digest it is pink Oxygen Test – obligate aerobes require oxygen, obligate anaerobes cannot grow in oxygen, facultative anaerobes can survive with or without oxygen (prefer 02), thiogylcollate removes O2, oxygen is only at surface, obligate aerobe will grow at top, obligate anaerobe will grow below surface, notmotile facultative anaerobes will grow at top and stab site, motile facultative anaerobes grow everywhere (turbid) Catalase Test – catalase enzyme degrades hydrogen peroxide producing water and O2 bubbles, some cells can degrade H2O2 because it can damage lipids and proteins, if bubbles bacteria is catalase positive R value – resolution value, minimum distance between objects needed so that they are visibly separate, smaller value means a better microscope R = 0.61l /N.A. 0.61 – relationship between condenser and objective lenses l of light (visible 400600 nm) N.A. – numerical aperture, relationship between refractive index of medium and size and working distance of the lens resolution value can be decrease by using smaller wavelength illuminator (electron microscope) or by increasing the N.A. (immersion oil medium deflects light less so more light enters objective lens) Empty Magnification – increasing magnification without increasing resolution
Lab 4 – Cytoskeleton: Cell Motility and Subcellular Movement Cytoskeleton – functions for cell structure, motility, organelle movement, biological processes, interact with many organelles
Microtubules – used for cell motility, flagella and cilia are made of microtubules9 outer doublets and 2 central, cilia can be used to feed by collecting food into oral groove and forming a food vacuole via phagocytosis, also responsible for organelle movement Microfilaments – amoeboid movement via pseudopodia, microfilaments assemble and disassemble to create viscous plasmagel and liquid plasmasol, plasmagel microfilaments interact with myosin to squeeze that part of the cell propelling plasmasol forward into extending pseudopodium, extendin pseudopodium stabilized by turning plasmasol into plasmagel, usually not responsible for organelle movement except in Paramecium where they move food vacuoles Intermediate Filaments – structural roles especially in the nuclear lamina of the nucleus, cells not undergoing mitosis have intact nuclear lamina Contrast – difference in intensity between object and medium stains can increase contrast, stains on living cells are called vital stains – allow observation of living functions for small time, fixed cells hold stain usually use a bright field microscope, if cells are transparent and medium is transparent cells appear invisible a phase contrast microscope takes advantage of the cell and medium’s different refractive index, slows light down and eye sees higher refractive indexes darker a fluorescence microscope lets you see glowing specimens under light, some cell structures need stain to fluoresce, others are autofluoresce, a dichroic mirror permits wavelengths emitted by the specimen Ingestion and digestion in Paramecium single celled protist – detain to slow movement, congo red dye infused yeast, when digested congo red in a good vacuole melds with a lysosome, when pH changes dye will turn yelloworange, move everywhere and spin, no one direction of movement by cilia, have an oralgroove, contractile vacuoles fill with water, red eyespot Swimming behaviour of Euglena single celled photosynthetic protist – vibrate back and forth in a forward motion, move in fairly straight line then switch, couldn’t see flagella, faster and more whip like than paramecium, red eyespot and lots of chloroplasts Pelomyxa carolinensis amoeba – move with pseudopodium, hard to see complete organism because it’s so thick, nonmoving plasmagel and streaming plasmosol towards pseudopodia, food vacuole algae fluoresce red spinach culture covered in bacteria and ciliates Proportion of Field – for microscopes without ocular micrometer Lab 5 – Amylase Enzyme Activity and Action of Inhibitors Assay – a test for a particular process, reaction or substance Enzymes – proteins (rarely RNAs) that catalyze metabolic reactions, facilitate reactions but are not consumed/destroyed, substratespecific, end in ase Substrate/Reactant – molecule to be metabolized Active Site – where in the enqyme the substrate binds as it is converted into an end product Activation Energy – energy required to initiate the change in the substrate, must be met for reaction to occur
Enzymes lower activation energy of a reaction and make it happen faster Amylose – component of starch, polymer of glucose monomers connected by alpha bonds Amylase – hydrolyses alpha bonds in amylose resulting in glucose monomers, dimmers (maltose) and shorter chains of amylose, useful for plants to break down stored starch from photosynthesis, in animals breaks down amylose so that it can be passed through cell membrane (humansin saliva and pancreas) Standard curve used to determine concentrations of amylose Colourimetric Assay – measures change in colour by measuring change in absorbance, iodine will react with starch and turn solution blue CarbBuster – marketed as a carb blocker, says it inhibits amylase from breaking down starch so that the body can’t absorb its calories Competitive Inhibitor – attaches in active site NonCompetitive Inhibitor – inhibits enzyme but doesn’t attach in active site Expect: amylase+amylose to be clear, amylose+amylose+carb buster to be blue, amylose+buffer to be blue, amylase+buffer to be clear, amylose+carb buster to be blue Measuring Enzyme Activity: perform assay… then transfer mixture every 3 minutes to test absorbance, use standard curve to determine the amylose quantity use linear region of graph to determine rate of reaction ex: (2 grams–14 grams )/(9 min–6 min) = 4 grams of amylose degraded per minute Absorbance may go up because of colour, or because of density/turbidity of having carb buster in it Carb buster actually worked on amylose doing something so that the iodine doesn’t turn it blue Lab 6 – Photosynthesis in Spinach Chloroplasts Chloroplasts – site of photosynthesis, reflect green and absorb other colours Chlorophyll – pigment that absorbs light in chloroplasts Photosynthesis – grouped into two sets of reactions, the light reactions and the Calvin cycle Light Reactions – series of reactions of energy transfer from light to chemical bonds Light energy strikes pigments in thylakoid membrane of chloroplasts Converted to electrical energy as excited electrons Chemical energy in the form of bonds in ATP of NADPH Photosystem II – light excites electrons in Chlorophyll P680 to electron transport chain, produces ATP Photosystem I – light excites electrons in chlorophyll P700 to electron transport chain, produces NADPH Calvin Cycle – uses ATP and NADPH in stroma to power fixation of carbon dioxide into sugar molecules, sideproducts NADP+ and ADP are recycled to light reactions DCPIP – indicator dye 2,6dichlorophenolindophenol, when oxidized turns blue, when reduced is colourless, accepts electrons from electron transport chain of Photosystem II reducing itself to clear
Absorption Spectrum – which wavelengths of light the pigments in chloroplasts maximally absorb, wavelenths maximally absorbed produce the highest rates of photosynthetic activity Shine light of a range of wavelengths (spectrophotometer) on a spinach (Spinacia oleracea) chloroplast suspension and measure absorbance Action Spectrum – indicates which colours of light produce highest rates of photosynthesis Measuring change in absorbance over 3 minute intervals when subjected to three different filter colours (determined by the reduction of the indicator DCPIP as it became less blue coloured as it accepted electrons from Photosystem II, use wavelength that oxidized blue DCPIP maximally absorbs independent variable – colour of light (so tested in dark too) Controls: DCPIP+buffer, chloroplasts+solvent(of DCPIP) Corrected Change in Absorbance=change in absorbance of DCPIP+chloroplasts – chloroplasts only – DCPIP only – dark control Light positioned at different distances because the filters have different density but you want constant light intensities Put beaker of cold water in front of filter so you don’t get heat from light Lab 7 – Sugar Metabolism in Yeast Cells need ATP which can be produced by catabolism of carbs, proteins and fats yeast Sacharomyces cerevisiae, singlecelled euk., metabolizes glucose by fermentation even in presence of oxygen Biofuels – fuels produced from biological substrates, trying to find optimal substrate to produce ethanol (instead of using petroleum) Want to produce as much ethanol with little substrate Corn is food and feed (so want to use nonedible parts) Corn syrup – glucose and fructose solution Kernel – starch, sugars and cellulose Stems – cellulose, hemicellulose, lignin Cobs – xylans, lignin, cellulose, little sugar amylase modifies starch, not cellulose Glycolysis – converts flucose to pyruvate, one molecule of glucose results in net production of 2 ATP via substratelevel phosphorylation, breakdown of pyruvate differs depending on presence/absence of oxygen Fermentation – a form of carbohydrate metabolism Breakdown of Pyruvate: In Presence of Oxygen – pyruvate acetyl CoA, moved into mitochondria and degraded by Krebs cycle 2 ATP/glucose by substratelevel phosphorylation, remaining ATP produced by oxidative phosphorylation, CO2 produced as by product (6/glucose) Absence of Oxygen – pyruvate converted to alcohol or lactic acid, alcohol fermentation produces ethanol and CO2
Alcohol Fermentation in Yeast – fermentation of glucose produces ethanol that can be toxic to yeast (1 glucose 2 ATP net, CO 2, 2 pyruvate > 2 acetaldehyde > 2 ethanol, 2 NAD+ Physical Response Curve – graphs that represent the reaction of a system to a stimulus, ex: carbon dioxide generate by alcohol fermentation to measure glucose metabolism directly, x access is time, y is physiological response, rate of reaction taken at linear log phase Lag – period of time required for reactions to begin (flat beginning) Log – reactions proceed at maximum rate (upsloping) Stationary – rate of reaction plateaus due to a lot of end products or lack of substrate/reagants (flat top) Death – quantity of dependent variable produced less than quantity consumed (downsloping) This experiment: durham tube filled with substrate/yeast mixture inverted to see how much CO2 collects from fermentation of glucose The test tubes are incubated at 30 degrees because yeast metabolizes sugars most effectively at this temperature (yeast left out to make sure reactions don’t occur yet) Swirl yeast to resuspend the yeast cells evenly Lab 8 – DNA Extraction Bacterial Genomic DNA – double stranded DNA helix arranged in a circle anchored to bacterial plasma membrane, ~4000 genes that encode all the functions of a bacterial cell Bacterial Plasmid DNA – floats freely in the cytoplasm, not all bacteria have it, usually circular in a supercoiled conformation in which circular double helix twists upon itself (both strands have to be intact), smaller than genomic DNA, ~225 genes not required for survival but give extra abilities to survive in some conditions plasmid DNA p107 – extracted from a strain of E. coli, can grow in presence of antibiotic kanamycin (will used this plasmid DNA to transform other cells in lab 9) Plasmids can be selected for if they confer advantages, but also are a disadvantage as there is more DNA to replicate Eukaryotic DNA – arranged in linear strands called chromosomes located in nucleus, humans23 pairs of chromosomes, human DNA ~3000035000 genes, large size High molecular weight DNA (HMW) Mini Plasmid Prep – obtaining small amount of plasmid DNA from bacteria, plasmid DNA is not very pure and also contains RNA (need maxi prep for purity and larger quantity), performed based on size and conformation of DNA molecules treatment with alkaline solution and neutralization then: plasmid small and supercoiled, so it stays in solution, larger genomic DNA will precipitate out from salmon sperm DNA: HMW has high affinity for glass so is easily extracted DNA solution in TE – a buffer Tris and EDTA to bind magnesium ions of DNAse so that it does not break down the DNA into nucleotides Circular Genomic DNA
Cellular Proteins Cell Wall/Plasma Membrane
RNA Supercoiled Plasmid DNA (p107) Microcentrifuge – creates centrifugal force, must be balanced with tubes on opposite sides, hinges pointing out so you know what side pellet collects on, liquid above pellet is supernatant Extracting pDNA Experiment: 1) Centrifuge E. coli for 20 sec. to get bacteria cells at bottom, vortex cells to disrupt pellet (so they can resuspend in next step) 2) STE Buffer is ice cold and washes any medium away from cells, vortex, centrifuge, pellet is left and vortexed 3) Solution I is ice cold, vortex to resuspend pellet, is buffered and isotonic 4) Solution II is room temperature, do not vortex (invert + flick) so genomic DNA doesn’t shear and contaminate plasmid DNA, contains sodium dodecysulfate (SDS) and alkali NaOH, detergent denatures proteins and disrupts plasma membrane, cell lyses and cell components go into solution, NaOH raises pH and denatures gDNA and 2 strands come apart but supercoiled plasmid does not come apart, tube very viscous because of gDNA, place on ice 5) Solution III is ice cold, invert not vortex, neutralizes pH because it’s acidic potassium acetate solution, put on ice, recreates Hbonds in gDNA into white precipitate (insoluble salt) and reforms correct Hbonds in pDNA, precipitate contains gDNA, cell wall debris, proteins…, centrifuge to get pellet of gDNA potassiumSDSproteincell wall complex out, pDNA remains in supernatant (into 2 new tubes) 6) 95% Ethanol and vortex, removes water, pDNA and RNA precipitate out, centrifuge, invisible pellet is RNA and pDNA 70% Ethanol and vortex, remove salt, hydrate pellet slightly so it can be dissolved, centrifuge, pellet is washed pDNA and RNA Incubate at 37 degrees for 30 min. to evaporate any ethanol leftover 7) Solution T added (Tris buffer) to tubes on ice, vortex to resuspend pellet and dissolve pDNA and RNA, kept on ice to make sure any leftover DNAse don’t break down pDNA Extraction of Eukaryotic HMW DNA: 1) Add sodium acetate solution to salmon sperm DNA, acts as a salt to help ppt out the HMW during ethanol ppt 2) 95% Ethanol, invert so DNA doesn’t shear, DNA ppt
3) remove DNA strands with sealed pipette, wash in 70% ethanol, let DNA dry 4) wash DNA off into Solution T, let pipette soak in it to make sure DNA is all off, tube now has pure HMW DNA in tris buffer Lab 9/10 – Transformation of Bacterial Cells Avery/MacLeod/McCarty Experiment – tested different macromolecules to see which would transform R type bacteria into harmful S type bacteria (Streptococcus pneumoniae), determined DNA holds genetic material, lysed S cells and added to R cells each time removing a macromolecule systematically: Proteins – chloroform extraction, proteases degraded them Polysaccarides – enzymatic digestion RNA – RNAse DNA – DNAse Selectable Marker – a marker you can use to select for or against a particular phenotype, ex: kanamycin resistance Experiment: test transformation in E. coli with pDNA, cells are competent to uptake DNA because they have been treated with CaCl2 (have holes in membrane), p107 extracted last week is kanamycin resistant, competent cells are kanamycin sensitive Add pDNA to cells: if they grow on kanamycin they have transformed Add DNAse to pDNA, then to cells: no growth without DNA because they have not transformed and are still k. sensitive Kanamycin – antiobiotic of aminoglycoside family, inhibit protein synthesis in prokaryotes Transcription – the use of a DNA template to synthesize RNA Translation – reading of mRNA to produce protein Phosphorylating – adding a phosphate group Transformation of cellskans to kanr: Without kan – cells grow normally, gDNA is transcribed to mRNA, ribosome binds mRNA and translates it to new proteins With kan antibiotic – kan enters cell by oxygen dependent active transport, kan binds to 30S small ribosomal subunit, mRNA cannot bind to ribosome, no new proteins, cell dies because it cannot grow with kan With kan and pDNA – p107 has a gene that is transcribed and translated to phosphotransferase enzyme which phosphorylates kanamycin making it inactive, cells grow with kan Plate Count Method – counts living cells only that have grown and divided to produce a colony (a viable cell count), concentration of viable cells in original culture is calculated with number of colonies on plate and dilution: Cu=Cd/D where C is number of colonies per volume plated, change to /mL (this will give you cells that were transformed) only agar plates with 30
Solvent Control Group – examines the solvent, tests that solvent doesn’t affect dependent variable 4. Draw a conclusion based on data obtained. Either refute or support your hypothesis (not proven). 5. Hypothesis to theory (after many test of a hypothesis) Osmosis – water movement across a semipermeable membrane, passive transport, water moves from an area of low solute to high solute concentration Cell survival depends on their ability to regulate influx and efflux of water Cell walls constrains size of cytoplasm, prevents membrane rupturing Hypotonic Solution – lower solute [ ] than cell cell swells – turgid Hypertonic Solution – higher solute [ ] than cell water leaves cell – plasmolysis Isotonic Solution – same [ ], no net mov’t of water reduced pressure – flaccid Marine algae should be able to handle higher [ ] salt solutions (Tetraselmis: motile, tiny, round) Fresh water algae should only be able to handle lower [ ] salt solutions before dying from plasmolysis (Mesotaenium: longer ovals) Lab 2 – Streaking Bacterial Cells, Membrane Structure and Function Membrane functions: separation from external environment, organization into organelles, and regulation of transport Role of structure studied by disrupting one or more of its functions Used temperatures to disrupt membrane function in Beta vulgaris Betacyanin – red pigment located in central vacuole Tonoplast – membrane surround central vacuole Plasma membrane – surrounded cell If these membranes damaged – betacyanin will leak out of cells and increase concentration of betacyanin in surrounding solution (measured indirectly and quantitatively by absorbance value) Spectrophotometry: Objects appear coloured because they absorb some of the visible spectrum and reflect the other wavelengths (cause their colour) Chromophore – part of a molecule that absorbs energy from light to excite its electrons, wavelength causing highest absorbance unique to chromophore (set spectrophotometer to specific wavelength of choromophore) Can create Absorption spectrums showing amount of light absorbed at different wavelengths Can measure concentration based on absorbance measurements Four parts: Light source is a tungsten filament lamp emitting white light, which is split into different colours by a prism, slit selects one wavelength of light, then the photoelectric tube compares incident light to transmitted light. Transmitted light:incident light = Transmittance (T), %age Absorbance (A) – negative log of transmittance, wavelength subscript Standard Curve – graph of absorbance and concentration, absorbancies measured with dilutions of a stock solution (known [ ]), specific to the pigment and buffer, cannot be
extrapolated (have to do a dilution of a solution with unknown concentration if its absorbance is beyond your standard curve) Dilution (D) = volume of original solution / volume of original + volume of solvent Cundiluted = Cdiluted / D Zero – set transmittance to zero with nothing in spectrophotometer Calibrate – set absorbance to zero with blank (all components of sample except molecule of interest) Optical Density – amount of light that is scattered or absorbed by particles suspended in solution, measures [ ] of cells in suspension Beet experiment: detailed in lab report Liquid Culture – suspension of millions of cells in a liquid medium with all nutrients required for growth Streaking – way to isolated single bacterial cells from liquid culture, 3 streaks each coming from each other, use aseptic technique (ex: flame inoculating loop), third streak will have isolated colonies of different types of bacteria Turbid – not clear, cloudy solution of for example, bacteria Lab 3 – Identification of Bacterial Species Identification of species based on: cell and colony morphology, chemical composition of cell walls, biochemical activities and nutritional requirements need pure culture by isolation (Streaking) GramPositive Bacteria – thick layer of peptidoglycan surrounding its one membrane, stain purple GramNegative Bacteria – thin layer of peptidoglycan, 2 phospholipid bilayers, outer membrane surrounding plasma/inner membrane, stain pink Gram stain – produces a colour result dependent of the cell wall structure of the bacteria, a differential stain dividing the bacteria into two groups 1. Stain – Crystal violet – picked up by cell wall of cells fixed to a slide, dyes peptidoglycan purple 2. Mordant – Iodine – increase the affinity of the dye to the peptidoglycan by forming large complexes with crystal violet, crystals in peptidoglycan 3. Decolorizing Agent – Ethanol – dissolves lipids in outer membrane of gram negative, wash off CVI complexes after 10 seconds, gramnegative turn colourless, the differential step (CVI will wash off grampositive but will take a longer time) 4. Counterstain – Safranin – pink/reddish dye that stains gramnegative, gram positive still purple GramPositive GramNegative Length of Exposure Crystal Violet Purple Purple 1 minute Gram’s Iodine Purple Purple 1 minute Ethanol Purple Clear 10 seconds Safranin Purple Pink 1 minute Colony Morphology – shape, size, edge, colour, surface texture, and elevation Cell Morphology – shape, arrangement, external structures, and gram stain Cocci – spherical
Bacilli – rod or cylinder shape, long and slender or short Spirilla – corkscrew shape arranged singly, in pairs, cluster, chains presence or absense of flagella, capsule, slime layer, fimbriae, pili Biochemical Test – many tests need to be performed to determine species of bacteria Motility Test – will swim away from area of injection to lower [ ] of bacteria using one or many flagella, the bacteria if they move will oxidize tetrazolium salt to red, if no movement will just be red along stab site Endospore Test – some grampositive and one gramnegative species can form endospores, endospores are highly resistant, dehydrated cells with thick walls and layers, can resist extreme temp., inoculate at 80 degrees and only endospores live, endospores is turbid, none is clear Glucose Fermentation Test – some bacteria metabolize glucose for their carbon energy source, medium with glucose and pH indicator phenol red will turn yellow if glucose is metabolized, if CO2 is made, bubbles will get trapped in inverted vial Tryptophanase Test – some bacteria metabolize the amino acid tryptophan for nitrogen, reaction is catalyzed by enzyme tryptophanase, end product reacts with DMCA and turns blue, if the cells don’t digest it is pink Oxygen Test – obligate aerobes require oxygen, obligate anaerobes cannot grow in oxygen, facultative anaerobes can survive with or without oxygen (prefer 02), thiogylcollate removes O2, oxygen is only at surface, obligate aerobe will grow at top, obligate anaerobe will grow below surface, notmotile facultative anaerobes will grow at top and stab site, motile facultative anaerobes grow everywhere (turbid) Catalase Test – catalase enzyme degrades hydrogen peroxide producing water and O2 bubbles, some cells can degrade H2O2 because it can damage lipids and proteins, if bubbles bacteria is catalase positive R value – resolution value, minimum distance between objects needed so that they are visibly separate, smaller value means a better microscope R = 0.61l /N.A. 0.61 – relationship between condenser and objective lenses l of light (visible 400600 nm) N.A. – numerical aperture, relationship between refractive index of medium and size and working distance of the lens resolution value can be decrease by using smaller wavelength illuminator (electron microscope) or by increasing the N.A. (immersion oil medium deflects light less so more light enters objective lens) Empty Magnification – increasing magnification without increasing resolution
Lab 4 – Cytoskeleton: Cell Motility and Subcellular Movement Cytoskeleton – functions for cell structure, motility, organelle movement, biological processes, interact with many organelles
Microtubules – used for cell motility, flagella and cilia are made of microtubules9 outer doublets and 2 central, cilia can be used to feed by collecting food into oral groove and forming a food vacuole via phagocytosis, also responsible for organelle movement Microfilaments – amoeboid movement via pseudopodia, microfilaments assemble and disassemble to create viscous plasmagel and liquid plasmasol, plasmagel microfilaments interact with myosin to squeeze that part of the cell propelling plasmasol forward into extending pseudopodium, extendin pseudopodium stabilized by turning plasmasol into plasmagel, usually not responsible for organelle movement except in Paramecium where they move food vacuoles Intermediate Filaments – structural roles especially in the nuclear lamina of the nucleus, cells not undergoing mitosis have intact nuclear lamina Contrast – difference in intensity between object and medium stains can increase contrast, stains on living cells are called vital stains – allow observation of living functions for small time, fixed cells hold stain usually use a bright field microscope, if cells are transparent and medium is transparent cells appear invisible a phase contrast microscope takes advantage of the cell and medium’s different refractive index, slows light down and eye sees higher refractive indexes darker a fluorescence microscope lets you see glowing specimens under light, some cell structures need stain to fluoresce, others are autofluoresce, a dichroic mirror permits wavelengths emitted by the specimen Ingestion and digestion in Paramecium single celled protist – detain to slow movement, congo red dye infused yeast, when digested congo red in a good vacuole melds with a lysosome, when pH changes dye will turn yelloworange, move everywhere and spin, no one direction of movement by cilia, have an oralgroove, contractile vacuoles fill with water, red eyespot Swimming behaviour of Euglena single celled photosynthetic protist – vibrate back and forth in a forward motion, move in fairly straight line then switch, couldn’t see flagella, faster and more whip like than paramecium, red eyespot and lots of chloroplasts Pelomyxa carolinensis amoeba – move with pseudopodium, hard to see complete organism because it’s so thick, nonmoving plasmagel and streaming plasmosol towards pseudopodia, food vacuole algae fluoresce red spinach culture covered in bacteria and ciliates Proportion of Field – for microscopes without ocular micrometer Lab 5 – Amylase Enzyme Activity and Action of Inhibitors Assay – a test for a particular process, reaction or substance Enzymes – proteins (rarely RNAs) that catalyze metabolic reactions, facilitate reactions but are not consumed/destroyed, substratespecific, end in ase Substrate/Reactant – molecule to be metabolized Active Site – where in the enqyme the substrate binds as it is converted into an end product Activation Energy – energy required to initiate the change in the substrate, must be met for reaction to occur
Enzymes lower activation energy of a reaction and make it happen faster Amylose – component of starch, polymer of glucose monomers connected by alpha bonds Amylase – hydrolyses alpha bonds in amylose resulting in glucose monomers, dimmers (maltose) and shorter chains of amylose, useful for plants to break down stored starch from photosynthesis, in animals breaks down amylose so that it can be passed through cell membrane (humansin saliva and pancreas) Standard curve used to determine concentrations of amylose Colourimetric Assay – measures change in colour by measuring change in absorbance, iodine will react with starch and turn solution blue CarbBuster – marketed as a carb blocker, says it inhibits amylase from breaking down starch so that the body can’t absorb its calories Competitive Inhibitor – attaches in active site NonCompetitive Inhibitor – inhibits enzyme but doesn’t attach in active site Expect: amylase+amylose to be clear, amylose+amylose+carb buster to be blue, amylose+buffer to be blue, amylase+buffer to be clear, amylose+carb buster to be blue Measuring Enzyme Activity: perform assay… then transfer mixture every 3 minutes to test absorbance, use standard curve to determine the amylose quantity use linear region of graph to determine rate of reaction ex: (2 grams–14 grams )/(9 min–6 min) = 4 grams of amylose degraded per minute Absorbance may go up because of colour, or because of density/turbidity of having carb buster in it Carb buster actually worked on amylose doing something so that the iodine doesn’t turn it blue Lab 6 – Photosynthesis in Spinach Chloroplasts Chloroplasts – site of photosynthesis, reflect green and absorb other colours Chlorophyll – pigment that absorbs light in chloroplasts Photosynthesis – grouped into two sets of reactions, the light reactions and the Calvin cycle Light Reactions – series of reactions of energy transfer from light to chemical bonds Light energy strikes pigments in thylakoid membrane of chloroplasts Converted to electrical energy as excited electrons Chemical energy in the form of bonds in ATP of NADPH Photosystem II – light excites electrons in Chlorophyll P680 to electron transport chain, produces ATP Photosystem I – light excites electrons in chlorophyll P700 to electron transport chain, produces NADPH Calvin Cycle – uses ATP and NADPH in stroma to power fixation of carbon dioxide into sugar molecules, sideproducts NADP+ and ADP are recycled to light reactions DCPIP – indicator dye 2,6dichlorophenolindophenol, when oxidized turns blue, when reduced is colourless, accepts electrons from electron transport chain of Photosystem II reducing itself to clear
Absorption Spectrum – which wavelengths of light the pigments in chloroplasts maximally absorb, wavelenths maximally absorbed produce the highest rates of photosynthetic activity Shine light of a range of wavelengths (spectrophotometer) on a spinach (Spinacia oleracea) chloroplast suspension and measure absorbance Action Spectrum – indicates which colours of light produce highest rates of photosynthesis Measuring change in absorbance over 3 minute intervals when subjected to three different filter colours (determined by the reduction of the indicator DCPIP as it became less blue coloured as it accepted electrons from Photosystem II, use wavelength that oxidized blue DCPIP maximally absorbs independent variable – colour of light (so tested in dark too) Controls: DCPIP+buffer, chloroplasts+solvent(of DCPIP) Corrected Change in Absorbance=change in absorbance of DCPIP+chloroplasts – chloroplasts only – DCPIP only – dark control Light positioned at different distances because the filters have different density but you want constant light intensities Put beaker of cold water in front of filter so you don’t get heat from light Lab 7 – Sugar Metabolism in Yeast Cells need ATP which can be produced by catabolism of carbs, proteins and fats yeast Sacharomyces cerevisiae, singlecelled euk., metabolizes glucose by fermentation even in presence of oxygen Biofuels – fuels produced from biological substrates, trying to find optimal substrate to produce ethanol (instead of using petroleum) Want to produce as much ethanol with little substrate Corn is food and feed (so want to use nonedible parts) Corn syrup – glucose and fructose solution Kernel – starch, sugars and cellulose Stems – cellulose, hemicellulose, lignin Cobs – xylans, lignin, cellulose, little sugar amylase modifies starch, not cellulose Glycolysis – converts flucose to pyruvate, one molecule of glucose results in net production of 2 ATP via substratelevel phosphorylation, breakdown of pyruvate differs depending on presence/absence of oxygen Fermentation – a form of carbohydrate metabolism Breakdown of Pyruvate: In Presence of Oxygen – pyruvate acetyl CoA, moved into mitochondria and degraded by Krebs cycle 2 ATP/glucose by substratelevel phosphorylation, remaining ATP produced by oxidative phosphorylation, CO2 produced as by product (6/glucose) Absence of Oxygen – pyruvate converted to alcohol or lactic acid, alcohol fermentation produces ethanol and CO2
Alcohol Fermentation in Yeast – fermentation of glucose produces ethanol that can be toxic to yeast (1 glucose 2 ATP net, CO 2, 2 pyruvate > 2 acetaldehyde > 2 ethanol, 2 NAD+ Physical Response Curve – graphs that represent the reaction of a system to a stimulus, ex: carbon dioxide generate by alcohol fermentation to measure glucose metabolism directly, x access is time, y is physiological response, rate of reaction taken at linear log phase Lag – period of time required for reactions to begin (flat beginning) Log – reactions proceed at maximum rate (upsloping) Stationary – rate of reaction plateaus due to a lot of end products or lack of substrate/reagants (flat top) Death – quantity of dependent variable produced less than quantity consumed (downsloping) This experiment: durham tube filled with substrate/yeast mixture inverted to see how much CO2 collects from fermentation of glucose The test tubes are incubated at 30 degrees because yeast metabolizes sugars most effectively at this temperature (yeast left out to make sure reactions don’t occur yet) Swirl yeast to resuspend the yeast cells evenly Lab 8 – DNA Extraction Bacterial Genomic DNA – double stranded DNA helix arranged in a circle anchored to bacterial plasma membrane, ~4000 genes that encode all the functions of a bacterial cell Bacterial Plasmid DNA – floats freely in the cytoplasm, not all bacteria have it, usually circular in a supercoiled conformation in which circular double helix twists upon itself (both strands have to be intact), smaller than genomic DNA, ~225 genes not required for survival but give extra abilities to survive in some conditions plasmid DNA p107 – extracted from a strain of E. coli, can grow in presence of antibiotic kanamycin (will used this plasmid DNA to transform other cells in lab 9) Plasmids can be selected for if they confer advantages, but also are a disadvantage as there is more DNA to replicate Eukaryotic DNA – arranged in linear strands called chromosomes located in nucleus, humans23 pairs of chromosomes, human DNA ~3000035000 genes, large size High molecular weight DNA (HMW) Mini Plasmid Prep – obtaining small amount of plasmid DNA from bacteria, plasmid DNA is not very pure and also contains RNA (need maxi prep for purity and larger quantity), performed based on size and conformation of DNA molecules treatment with alkaline solution and neutralization then: plasmid small and supercoiled, so it stays in solution, larger genomic DNA will precipitate out from salmon sperm DNA: HMW has high affinity for glass so is easily extracted DNA solution in TE – a buffer Tris and EDTA to bind magnesium ions of DNAse so that it does not break down the DNA into nucleotides Circular Genomic DNA
Cellular Proteins Cell Wall/Plasma Membrane
RNA Supercoiled Plasmid DNA (p107) Microcentrifuge – creates centrifugal force, must be balanced with tubes on opposite sides, hinges pointing out so you know what side pellet collects on, liquid above pellet is supernatant Extracting pDNA Experiment: 1) Centrifuge E. coli for 20 sec. to get bacteria cells at bottom, vortex cells to disrupt pellet (so they can resuspend in next step) 2) STE Buffer is ice cold and washes any medium away from cells, vortex, centrifuge, pellet is left and vortexed 3) Solution I is ice cold, vortex to resuspend pellet, is buffered and isotonic 4) Solution II is room temperature, do not vortex (invert + flick) so genomic DNA doesn’t shear and contaminate plasmid DNA, contains sodium dodecysulfate (SDS) and alkali NaOH, detergent denatures proteins and disrupts plasma membrane, cell lyses and cell components go into solution, NaOH raises pH and denatures gDNA and 2 strands come apart but supercoiled plasmid does not come apart, tube very viscous because of gDNA, place on ice 5) Solution III is ice cold, invert not vortex, neutralizes pH because it’s acidic potassium acetate solution, put on ice, recreates Hbonds in gDNA into white precipitate (insoluble salt) and reforms correct Hbonds in pDNA, precipitate contains gDNA, cell wall debris, proteins…, centrifuge to get pellet of gDNA potassiumSDSproteincell wall complex out, pDNA remains in supernatant (into 2 new tubes) 6) 95% Ethanol and vortex, removes water, pDNA and RNA precipitate out, centrifuge, invisible pellet is RNA and pDNA 70% Ethanol and vortex, remove salt, hydrate pellet slightly so it can be dissolved, centrifuge, pellet is washed pDNA and RNA Incubate at 37 degrees for 30 min. to evaporate any ethanol leftover 7) Solution T added (Tris buffer) to tubes on ice, vortex to resuspend pellet and dissolve pDNA and RNA, kept on ice to make sure any leftover DNAse don’t break down pDNA Extraction of Eukaryotic HMW DNA: 1) Add sodium acetate solution to salmon sperm DNA, acts as a salt to help ppt out the HMW during ethanol ppt 2) 95% Ethanol, invert so DNA doesn’t shear, DNA ppt
3) remove DNA strands with sealed pipette, wash in 70% ethanol, let DNA dry 4) wash DNA off into Solution T, let pipette soak in it to make sure DNA is all off, tube now has pure HMW DNA in tris buffer Lab 9/10 – Transformation of Bacterial Cells Avery/MacLeod/McCarty Experiment – tested different macromolecules to see which would transform R type bacteria into harmful S type bacteria (Streptococcus pneumoniae), determined DNA holds genetic material, lysed S cells and added to R cells each time removing a macromolecule systematically: Proteins – chloroform extraction, proteases degraded them Polysaccarides – enzymatic digestion RNA – RNAse DNA – DNAse Selectable Marker – a marker you can use to select for or against a particular phenotype, ex: kanamycin resistance Experiment: test transformation in E. coli with pDNA, cells are competent to uptake DNA because they have been treated with CaCl2 (have holes in membrane), p107 extracted last week is kanamycin resistant, competent cells are kanamycin sensitive Add pDNA to cells: if they grow on kanamycin they have transformed Add DNAse to pDNA, then to cells: no growth without DNA because they have not transformed and are still k. sensitive Kanamycin – antiobiotic of aminoglycoside family, inhibit protein synthesis in prokaryotes Transcription – the use of a DNA template to synthesize RNA Translation – reading of mRNA to produce protein Phosphorylating – adding a phosphate group Transformation of cellskans to kanr: Without kan – cells grow normally, gDNA is transcribed to mRNA, ribosome binds mRNA and translates it to new proteins With kan antibiotic – kan enters cell by oxygen dependent active transport, kan binds to 30S small ribosomal subunit, mRNA cannot bind to ribosome, no new proteins, cell dies because it cannot grow with kan With kan and pDNA – p107 has a gene that is transcribed and translated to phosphotransferase enzyme which phosphorylates kanamycin making it inactive, cells grow with kan Plate Count Method – counts living cells only that have grown and divided to produce a colony (a viable cell count), concentration of viable cells in original culture is calculated with number of colonies on plate and dilution: Cu=Cd/D where C is number of colonies per volume plated, change to /mL (this will give you cells that were transformed) only agar plates with 30
