lOMoARcPSD|24764822 BIOL 107 Lab 1-8 Study Notes for Lab Exam Introduction to Cell Biology (University of Alberta) Studocu is not sponsored or endorsed by any college or university Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 BIOLOGY 107 LAB FALL SEMESTER 2018 Pink - Vocabulary Red - Important Information Blue - Formulas Orange - Important Words Lab Quiz #3 (In-Class) - Study Notes LAB 1. INTRODUCTION, MICROSCOPES, SCIENTIFIC METHOD, AND OSMOSIS ● Pure Culture - an isolated group of genetically identical cells ○ Can be easily contaminated with another cell type because microorganisms are everywhere in the environment (including air) ○ Reduce chances of contamination by following a s terile (aka a septic) technique ● Colony - a small mass of genetically identical cells ○ Produced by microorganisms ability to grow and divide multiple times ○ Contains millions or billions of cells ● Medium - a solution or solid that contains nutrients cells can use to grow and divide ○ The solid medium in this lab is the a gar plate ● Lawn of Bacteria - a dense, even layer of bacteria on a medium Introduction to the Microscope ● Three types of lenses: 1. The Condenser Lens - focuses the light on the specimen 2. The Objective Lens - magnifies the image 3. The Ocular Lens - magnifies and inverts the image for viewing Brightfield (Light) Microscope Parts: 1. Light Source - a small, powerful light bulb 2. Condenser Lens - focuses the light onto the specimen a. The condenser focusing knob moves the condenser lens to focus it 3. Field Iris Diaphragm - controls the amount of light entering the lens system 1 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 4. 5. 6. 7. 8. a. Decreasing the amount of light can improve contrast (the difference between light and dark areas on the slide) making it easier to distinguish specimens b. Also used when the condenser lens is focused Stage - the horizontal surface where the slide is placed a. Moves horizontally to locate an object on the slide b. Moves vertically to focus the slide Coarse Focus Knob - moves the stage up and down to bring the specimen into focus Objective Lens - gathers light from the specimen, magnifies the images, and projects the magnified image into the body tube Fine Focus Knob - small knob on the coarse focus knob a. Brings the image into sharper focus under low power b. Used for all focusing when using high power lenses Ocular Lens - magnifies the images a. Aka the e yepiece b. Contains a measuring scales called an o cular micrometer 9. ● Objective lenses of varying magnification are mounted on a rotating nosepiece Microscopes Magnify and Resolve 2 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 ● Magnifying an object increases its apparent size ● Total magnification of the microscope is the product of the magnification of the objective lens and the ocular lens ● Resolution is the capacity to distinguish two adjacent points as being distinct ● Resolution value (R) is the minimum distance required between two points for them to remain visibly identifiable as separate points ● Calculating total magnification of a microscope formula: Total Magnification = Magnification Ocular lens x MagnificationObjective Lens Determining the Actual Size of a Specimen ● Two Methods: 1. Proportion of Field of View Occupied by the Specimen ○ Can approximate the size of the specimen by estimating the proportion of the field of view occupied by the specimen Objective Lens Magnification Field Diameter (mm) 10X 2.0 40X 0.5 100X 0.05 ○ Formula Size of Cell = Field diameter x Length of Cell(Under the lens) 2. Ocular Micrometer ○ Ocular micrometer - the miniature ruler set in one of the ocular lenses of the microscope ○ Must be calibrated with a s tage micrometer (a microscopic ruler with known measurements) because it does not have any values or units assigned to the gradations on the scale ○ Ocular micrometer divisions ○ Measure the cell using the ocular micrometer and multiply the length by the length of each ocular division at that magnification Objective Lens Magnification Ocular Micrometer Calibration ( μm/division) 10X 10 40X 2.5 3 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 100X 1 ○ Formula: Size of Cell = Ocular Divisions x Ocular Micrometer Calibration Calculating Magnification of Drawing ● Magnification - the ratio of the size of the magnified image to the actual size of the object ● Formula: Magnification of Drawing = Size of the Drawing Actual Size of the Specimen ● The size of the drawing is determined by measuring with a ruler ● The actual size of the specimen is determined using the proportion of field method or using the ocular micrometer ● IMPORTANT: the measurement units of the size of the drawing and the actual size of the specimen must be converted so that the units match ○ Both must be in m mo r c m or μ m Osmosis ● Cell survival is dependent on the cell’s ability to regulate the influx and efflux of water through its membranes ● Water moves across membranes from an area of low solute concentration to an area of high solute concentration ● Osmosis - movement of water across a semi-permeable membrane ○ Passive t ransport ● Hypotonic Solution - a solution that has a l ower solute concentration than the cell cytoplasm ○ The cell wall constrains the size of the cytoplasm and prevents the plasma membrane from rupturing when the cell wall is place in a hypotonic solution ○ The cell will fill with water and swell, becoming t urgid ● Hypertonic S olution - a solution that has a h igher solute concentration than the cell cytoplasm ○ Water will leave the cell and the plasma membrane will pull away from the cell wall (plasmolysis) ○ Eventually the cell will die ● Isotonic S olution - a solution that has the a s olute concentration the same as the cell 4 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 ○ There is no net movement of water, this causes the cell to become flaccid because of reduced pressure on the cell wall The Scientific Method ● Hypothesis format: i f, then, because ○ Ex. if you do X, then Y will happen, because of published knowledge that says Z. ○ Test the hypothesis by observation, experimentation, or both ● Experiments compare two treatments that vary by one variable only - the manipulated variable ● The two treatment groups are the control group and the experimental group ○ Control group - where the manipulated variable is absent ○ Experimental group - where the manipulated variable is present ● All other aspects of the experiment must be the same between the control and experimental groups ○ Ex. temperature, time, concentrations, etc ■ These are called c onstants ● The main assumptions of experiments is that the manipulated variable affects the r esponding variable ● Solvent control group - a control group that examines the solvent used in the experiment ○ Confirms that the effect on the responding variable is due only to the responding variable, and not the solvent it is dissolved in 5 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 ● Positive and negative control groups - control groups that are designed to give positive or negative results to test the assumptions behind the experimental components ● Controlled variables - regimes/conditions that are applied to both the control and experimental groups ○ Used to measure the effects of the manipulated variable on the responding variable under different conditions ● **avoid saying anything is proven; all that can be said is that the current evidence supports the hypothesis LAB 2. MEMBRANE STRUCTURE AND FUNCTION Introduction - Membranes and Summary of the Lab ● Some key functions: ○ Separation of the cell contents from the external environment ○ Organization of chemicals and reactions to specific organelles ○ Regulation of the transport of certain molecules into and out of the cell and its organelles ● Lab: focuses on using temperature to disrupt membrane function in living beet cells (Beta vulgaris) ○ Beet cells’ red pigment (betacyanin) is found in the cell’s large central vacuole which is surrounded by a membrane called the t onoplast ○ The cell itself is surrounded by the p lasma membrane ○ As long as the tonoplast and the plasma membrane remain intact, the betacyanin will remain inside the cell ○ If the two membranes are damaged, the betacyanin will leak out of the cell and cause the water to change color (from clear to red) ○ The s pectrophotometer is used to quantitatively measure the amount of betacyanin in the solution General Principles of Spectrophotometry ● An object appears colored because it absorbs part of the visible spectrum and reflects or transmits the other wavelengths ● Chromophores - molecules or parts of molecules that absorb light ● A s pectrophotometer can be set to measure the absorbance of a chromophore at the wavelength that a chromophore maximally absorbs, or it can be used to measure absorbance over a series of wavelengths ● The spectrophotometer can be used to: 1. Characterize absorbance patterns of solutions or pigments a. An absorption graph is a graph that shows the amount of light absorbed by a chromophore at a number of wavelengths b. When used in this way, the spectrophotometer can identify molecules 6 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 2. To measure concentration a. Concentration is proportional to absorbance ● The spectrophotometer has a white light that passes through a prism that separates it. A narrow slit selects the wavelength that passes through the sample and a photoelectric tube reads the light passing through the sample and compares the incident light and the transmitted light. ○ The ratio of transmitted light to incident light is called t ransmittance (T), which is expressed as a percentage ○ The negative log of transmittance is called a bsorbance (A) ■ The absorbance of a molecule has no units, but specifies the wavelength that was used ● Absorbance is a linear function of solute concentration ● Transmittance is not linearly related to concentration and is therefore not used to represent the amount of energy absorbed by a chromophore ● Particles in suspension scatter light (turbidity) rather than absorb it ● Both light absorption and light scattering prevent light from being transmitted ● The absorbance scale of the meter is used to determine the amount of light that is scattered or absorbed ○ This measurement is reported as optical density, or ODxxx, where the subscript indicated the wavelength of light Creating and Using a Standard Curve ● Stock solution - a concentrated solution of a solute in a known concentration ● Standard curve - a graph that shows the relationship between absorbance and concentration ● The absorbance value of a pigment solution is directly proportional to its concentration within a given range ○ Absorbance values of samples with unknown concentrations can then be compared to the standard curve to determine their concentrations Rules for Using a Standard Curve 1. A standard curve is specific to the pigment and its buffer 2. A standard curve cannot be extrapolated; cannot use an absorbance that is beyond the range of the standard curve to calculate a concentration ○ To measure the concentrations of a sample that has an absorbance beyond the range of the standard curve, a dilution must be done 7 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 ○ Using the diluted sample, the concentration of the original sample can be calculated 3. All samples measured must be in the same solvent or media as the standard curve ○ The absorbance scale of the spectrophotometer must be calibrated to 0.0 using a blank ○ The blank contains all the components of the sample being measure, except the molecule of interest to ensure that only the absorbance of the molecule is measured and not the solvent/media ○ The blank is usually the solvent/media the molecule is dissolved in or kept in ■ Ex. the beets were kept in water, so the water is used as a blank ● Formulas: of the Original Sample 1. Dilution (D) = V olume of Vtheolume Original Sample + V olume of Solvent 2. Cu = CD ÷ D Cu = concentration of undiluted (original) sample CD = concentration of the diluted sample D= Dilution LAB 3. CELL PHONE BIOLOGY AND ANTIBIOTIC RESISTANCE ● Microbiota - the types of bacteria growing in or on our bodies ○ Aka. flora ● The growth patterns will show how common items can be a source of contamination, or what can become contaminated ● Following a septic technique (aka s terile technique) when culturing microorganisms will help: ○ Prevent bacteria in the environment from contaminating your work’ ○ Prevent bacteria in your work from contaminating you and the environment ● Aseptic Technique ○ Sterilize the surfaces in your area to kill bacteria and prevent them from contaminating your cultures ■ Wash hands, wash work areas, flame items by passing them through a bunsen burner, glassware and mediums to be sterilized with high temperatures and pressure in an a utoclave ○ Reduce the exposure time ○ Work in an area with a low resident population of bacteria ● 8 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 ● Zone of inhibition - an area of clearing around the disc where sensitive organisms are unable to grow because they are sensitive to the antibiotic in the disc ● If the organisms in the culture are antibiotic resistant, there will be no zone of inhibition ● What are antibiotics? ○ Substances that prevent or inhibit bacterial and/or fungal growth ○ Can be broad spectrum and act against a variety of bacteria ○ Can be specific to a narrow range of bacteria ● Where do antibiotics come from? ○ Many of the first antibiotics that were discovered were produced by microorganisms ○ Used as a sort of biological warfare between competing species, between organisms of the same species to communicate, in a symbiotic way to support organisms of a different species, or may be intermediates of metabolism ○ Antibiotic resistance - the ability to actively avoid the actions of an antibiotic ● How does antibiotic resistance spread? ○ Two ways: 1. Mutation of bacterium’s genes 2. Horizontal transfer from another organism ○ In a mutation, several small changes enable one organism of the population to become resistant or tolerant of the antibiotic ○ In horizontal transfer, one organism passes genes for antibiotic resistance or tolerant of the antibiotic ■ These genes are often found on small circular DNA molecules called p lasmids ● A small dose or an inadequate dose will eliminate the bacteria that are sensitive to the antibiotic, leaving only those that are tolerant or resistant to it ● What are mechanisms of resistance? ○ Cells can modify the antibiotic itself via degradation or alteration ○ Cells can block influx (how the antibiotic enters the cell) or cause efflux by pumping it out ○ The target of the antibiotic can be protected or removed ● Tetracycline ○ Broad spectrum antibiotic ○ Acts against a variety of bacteria that can make people ill ○ Acts by preventing bacterial protein synthesis ● Penicillin ○ Generally used to treat G ram-positive bacteria ○ Acts by inhibiting crosslinking in the bacterial cell wall 9 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 ● Chloramphenicol ○ Broad spectrum antibiotic ○ Because of widespread antibiotic resistance, used only for pink eye in North America ○ Acts by inhibiting bacterial protein synthesis cxxc L ab Quiz #8 (In-Class) - Study Notes LAB 6. PHOTOSYNTHESIS AND CELLULAR RESPIRATION IN SPINACH ● There are two two major metabolic processes 1. Cellular Respiration 2. Photosynthesis Cellular Respiration ● A process that consumes oxygen and glucose and forms carbon dioxide and ATP ● An absence of oxygen in packaging of produce can lead to cellular death and decomposition in produce ● Produces a lot of ATP and it is the main way the cell powers itself ● If there is no light, ONLY cellular respiration can occur ● Formula: ○ Glucose + O2 = CO2 + ATP + H2O The Three Phases of Cellular Respiration 1. Glycolysis (cytoplasm) ○ Glucose is hydrolyzed in a multistep process to form pyruvate ○ ATP and NADH are produced 2. Pyruvate is oxidized and the product enters the citric acid cycle (mitochondria) 10 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 ○ Citric acid cycle is an eight step cycle that produces ATP, NADH, FADH2, and CO2 3. Oxidative Phosphorylation ○ NADH and FADH2 transfer their electrons to a chain of proteins and carriers called the electron transport chain (inner membrane) ○ The electrons are passed down the chain, releasing energy which is used to pump protons to make ATP ○ At the end of the chain, the electrons are transferred to oxygen, forming water ○ This is where the bulk of ATP production occurs Photosynthesis ● Takes light energy, carbon dioxide, and water to produce oxygen and glucose ● In all aerobic photosynthetic organisms these processes can happen at the same time in the same cells ● If there is no light, photosynthesis will NOT occur ● Formula: ○ H2O + light + CO2 = glucose + O2 The Two Phases of Photosynthesis 1. In the light reactions (thylakoid membranes) light excites pigments in photosystem II, causing a loss of an electron ○ The electron is passed along an electron transport chain, releasing energy which is used to pump protons to make ATP ○ At the end of the chain, the electrons are transferred to NADP+, forming NADPH ○ The electron ‘hole’ left in photosystem II is filled by lysing water, releasing O2 2. Calvin Cycle (stroma) ○ Uses ATP and NADPH to incorporate/fix CO2 into glucose ● The change in carbon dioxide gas in a seal vessel as a spinach leaf sits in the dark will also be used as a direct measurement of the rate of cellular respiration. ○ The greater the production of carbon dioxide, the greater the respiration rate 11 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 ● The change in percent oxygen gas in a sealed vessel as a spinach leaf sits in the dark will be used as a direct measurement of the rate of cellular respiration ○ The greater the consumption of oxygen, the greater the respiration rate ● The change in percent gas in a seal vessel during exposure to different intensities of light will be used as a direct measurement of the rate of photosynthesis ○ The greater the production of the oxygen, the greater the photosynthetic rate ● A direct assay measures the production of an end product or the consumption of a substrate ● An indirect assay measures a secondary compound as a proxy for the process or product of interest (ie. change of color) ● In bright light, spinach leaves will produce oxygen and consume carbon dioxide ● Oxygen will increase overall and carbon dioxide will decrease overall ● In the dark, spinach leaves will consume oxygen and produce carbon dioxide ● Oxygen will decrease overall and carbon dioxide will increase overall Describing The ‘Middle’ of a Data Set ● The middle of the data set is called the mean, which is also known as the average ○ The total of the values divided by the number of measurements ● The median can be determined by lining ip the values from highest to lowest, then determining what the very middle value is ○ Also called the 50th percentile: the value at which 50% of the data points are below Assessing Variance of a Data Set ● A histogram can be used to show the distribution of data ○ Plots the frequency of each value against the value itself ○ Often show the frequencies of ranges of values ● Distribution within a data set is commonly shown as standard deviation (SD) ○ Method used to report variance ○ Gives you a quantitative way to describe the distribution of the data 12 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 ○ The SD tells that approximately ⅔ of all sample data lie from one SD below the mean to one SD above it ○ Generally, in a cell biology experiment, you want the smallest standard deviation possible ○ If the SD is too high, it means there is a large spread in your data which could indicate a problem with the experiment or it could mean that your hypothesis is incorrect ○ Formula: Distance to mean: − mean Z= data point SD Standard Deviation: What it represents, and how to report it on a graph 1. If a graph gives the standard deviation, you have a better idea of how scattered the data is. The 68-95-99.7 rule says that 68%, 95%, and 99.7% of a sample are within one, two, and three standard deviations from the mean 2. Standard deviation also represents a unit of measure to describe the distance of a data point from the mean, namely the Z score a. For example, if a student scored 60/80 on the exam, you could calculate how many standard deviations they are above the mean i. Z = (60 - 47.8)/(13.8) = 0.88 SD from the mean 3. In some cases, researchers compare samples of two different populations or two different treatments in an experiment LAB 7. SUGAR METABOLISM IN YEAST ● Corn kernels contain starch, sugars, and cellulose ● Stems contain cellulose, hemicellulose, and lignin and may contain a little sugar or starch ● Cobs contain xylans, lignin, cellulose, and very little sugar ● Cells require ATP to survive ● ATP can be produced through the catabolism of carbohydrates, proteins, and fats ● Fermentation ○ A form of carbohydrate metabolism that allows cells to produce ATP ● Yeast is a single-celled eukaryotic organism; this strain of yeast is significant because it metabolizes glucose using fermentative pathways, even in the presence of oxygen 13 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 Glucose Metabolism in Eukaryotes ● The catabolism of glucose in eukaryotes begins with cytosolic reactions of glycolysis to convert glucose to pyruvate ● During these reactions one molecule of glucose results in the net production of 2 molecules of ATP via substrate-level phosphorylation ● The reactions of glycolysis occur whether or not oxygen is present ● The subsequent breakdown of pyruvate differs depending on the presence or absence of oxygen Pyruvate Metabolism in the Presence of Oxygen ● Most eukaryotes convert pyruvate into acetyl CoA, which is transported into the mitochondria and further degraded by the reactions of the citric acid cycle ● The reactions of the citric acid cycle produce ATP via substrate-level phosphorylation and oxidative phosphorylation use the energy from NADH and FADH2 to produce more ATP ● 6 molecules of CO2 are produced for every molecule of glucose metabolized Pyruvate Metabolism in the Absence of Oxygen ● The most common pathways to degrade pyruvate in the absence of oxygen are alcohol fermentation and lactic acid fermentation ● Fermentation regenerate NAD=, a key component required for the continuation of the glycolytic pathway ● Without a source of NAD=, glycolysis will cease, ATP production would cease, and the cell would eventually die ● Alcohol fermentation results in the production of ethanol ○ Used in biofuel, industrial, pharmaceutical, and other applications ● Although fermentation of glucose produces high ethanol, high concentrations of ethanol are toxic to yeast ● Carbon dioxide generated by alcohol fermentation will be used to generate a physiological response curve ○ These curves can show metabolism or growth in the presence or absence of a toxin like an antibiotic or a nutrient like sugar ○ Can be used to determine the effects of varying doses of a substance, called a dose-response curve ○ The rate is measured during the linear portion of the log phase of the curve 14 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com) lOMoARcPSD|24764822 LAB 8. DNA EXTRACTION Bacterial Genomic DNA ● Encode all the essential function of a bacterial cell (approx. 4000 genes in total) ● Arranged in a circle that is anchored to the bacterial plasma membrane Bacterial Plasmid DNA ● Present in some, but not all, bacterial cells ● Plasmids are usually small, circular, and float freely in the cell’s cytoplasm ● Usually in a supercoiled conformation, where the circula double helix molecule becomes twisted upon itself ● Supercoiling can only occur in a circular molecule in which both DNA strands are intact (no breaks in the DNA) ● The genes located on a plasmid are not required for survival of the cell when it is in optimal living conditions ○ However, some plasmid genes can confer extra attributes to the cell that allow the cell to survive in condition it could not survive in without the plasmid genes ■ These include genes involved in catabolism of unusual compounds, as well as resistance or virulence genes that allow colonization of an otherwise unsuitable environment Eukaryotic DNA ● Arranged in linear strands called chromosomes that are located in the nucleus of the cell ● Humans have 23 pairs of chromosomes or 46 chromosomes total ● Each chromosome is approximately 2x108 base pairs long and the amount of DNA in the entire human genome is approximately 3.2x109 base pairs ● Because of its large size, eukaryotic DNA is often referred to as high molecular weight (HMW) DNA 15 Downloaded by Aditi Patel (aditi2004.stkabir@gmail.com)
