Biol3460 Lab 1 1 Biology 3460 - Plant Physiology - Lab Exercise 2 Basic Lab Techniques Objectives: This lab is intended to: (1) provide an introduction to basic techniques used in biology labs (2) provide some experience with the equipment employed in future labs. Part A. Microscopy The compound light microscope is a very important tool in the study of biology. Typical compound light microscopes allow specimens to be viewed at 400 to 1000 times their actual size (total magnification is determined by multiplying the objective lens magnification by the ocular lens magnification). The light microscope is made up of a system of lenses aligned in such a way that allows magnification, as well as improved resolution. The resolution obtainable is also dependent on contrast in the specimen. Contrast can be adjusted with the iris diaphragm, located below the condenser lens, and can also be enhanced by the addition of stains to the specimen. Scientific drawings of specimens observed under the light microscope are often used to provide a record of what was viewed. Proper scientific drawings should be neatly drawn, with smooth lines, in pencil (they should never be in colour). More than one cell should be drawn to illustrate the cellular association to the observer (e.g. found singly or as part of a tissue). Labels should be added using straight horizontal lines to the right side of the drawing, and care should be taken with singular and plural forms of structural terminology. To demonstrate differences in density of specific structures, use stippling rather than shading. A figure caption that indicates the specimen viewed, how viewed, and the magnification of the drawing is always necessary. Practice your microscopy technique by viewing slides of Elodea, spinach leaf, and pea seeds. Work in pairs to set-up and view each slide preparation. 1. Place one leaflet of Elodea on a slide with a drop of water and look for the clearly visible chloroplasts. You should notice that the chloroplasts are circulating around in each cell. What is the purpose of this cytoplasmic streaming? 2. Prepare a slide of spinach leaf epidermis by peeling back a bit of the undersurface of the leaf tissue. You should get a thin section of only the clear epidermal tissue, leaving behind the parenchyma cells of the leaf itself. Find the guard cells and the stomata and make a labeled sketch of a section of epidermal tissue. Use an ocular micrometer to determine the actual size of the guard cells and then calculate the drawing magnification (drawing size actual size) for your figure and include it in your figure caption. 3. Slice a very thin section of imbibed pea seed tissue and place it on a slide with a drop of iodine stain. What do you think the bulk of the pea seed is composed of? What compound is commonly stored by plants as a food reserve?
Biol3460 Lab 1 2 Part B. Analytical Balance and Weighing Accuracy Careful use of all types of equipment is essential to obtaining accurate and repeatable results in a biology lab. In this exercise you will practice use of the balance, as well as perform a mini experiment that indicates the effect of imbibition on bean seeds. Care and use of the balance: Avoid shock and vibrations. Make sure the balance is level using the leveling bubble and the adjustable legs. Always use a weigh boat or weighing paper. Do not place any material directly on the balance pan. Use a damp paper towel to wipe up any spills and keep the pan and case clean at all times. Work in a group of four to complete this exercise. One pair of students should weigh the imbibed seeds then the other pair should weigh the dry seeds. 1. Place a weigh boat on the balance and press tare. 2. Gently blot each seed on a piece of paper towel. 3. Place the bean seed in the weigh boat and record its weight in Table 1. 4. Calculate the means and standard deviations for the weights of the dry and the imbibed seeds. 5. Perform a t-test on the data to determine if there is a significant effect of imbibition on bean seed weight. 6. What is the level of accuracy of the balance? Table 1. Weight of dry and imbibed bean seeds determined using analytical balance. Treatment Bean Seed Weights (g) Dry Imbibed Part C. A Bit of Chemistry and Solution Dilutions A basic understanding of general chemistry is very important to many topics in plant physiology. In the lab setting, in particular, it is necessary to be able to accurately prepare solutions of known concentrations and prepare dilutions from stock solutions. This exercise will give you practice with the preparation of solutions of various concentrations, as well as practice using pipettes to transport accurate volumes between solutions. Work in a group of four to complete the dilution series. 1. Obtain 7 test tubes from the side bench and label them with the appropriate protein standard concentrations (see Table 2). 2. Beginning with the 5mg/mL bovine serum albumin (BSA) stock solution, prepare the series of dilutions as outlined in Table 2. You will be responsible for calculating how much of each solution is required to prepare the next dilution in the series. 3. Prepare each solution by pipetting the appropriate amount of the previous concentration into the correctly labeled test tube and adding the appropriate volume of distilled water. 4. Mix each solution on the vortex for 15 seconds. 5. Use these solutions to continue with the procedure in Part D.
Biol3460 Lab 1 3 Table 2. Volume of protein solutions and distilled water to make a dilution series for protein analysis and standard curve construction. Protein concentration (mg/ml) Volume of protein solution (ml) Volume of water (ml) Total volume (ml) 1.0 ml of 5.0 mg/ml stock 6.0 0.75 ml of 1.0 mg/ml solution 6.0 0.50 ml of 0.75 mg/ml solution 6.0 0.25 ml of 0.50 mg/ml solution 6.0 0.10 ml of 0.25 mg/ml solution 6.0 0.05 ml of 0.10 mg/ml solution 6.0 0.0 6.0 Part D. Protein Assay and Spectrophotometer Use Many plant molecules such as the photosynthetic pigments, chlorophylls, anthocyanins, and carotenoids, naturally absorb light. Other colorless plant molecules can be converted to light-absorbing pigments by the addition of specific chemicals. A spectrophotometer is an instrument that can be used to objectively quantify the amounts and wavelengths of light that are absorbed by molecules in a solution. To practice using the spectrophotometer, you will conduct a Bradford protein assay using the solutions you prepared in Part C. Since protein is not highly coloured it can be reacted with another reagent to produce a coloured compound. Protein concentration is a useful measure to express various processes studied and allows comparison to other samples and to published values. Work as a group of four for this exercise. 1. Label 7 small tubes with the concentrations listed in Table 2, plus 2 test tubes with Unknown A and Unknown B. 2. Pipette 0.1mL of each protein solution into the appropriately labeled tube. 3. Add 5.0mL of the Bradford Reagent and vortex to mix. 4. Allow tubes to incubate for five minutes then use the spectrophotometer to read the absorbances at 595 nm. Spectrophotometer Procedure: 1. Set the wavelength to the appropriate value. 2. With no cuvette in the spectrophotometer set the spectrophotometer to zero on the percent transmittance scale, using the left-hand knob. 3. Always wipe the cuvette with a Kimwipe before inserting it into the spectrophotometer, to remove any dust, fingerprints, or water droplets that may affect how the sample absorbs light. 4. Place the reagent blank in the spectrophotometer and set to zero on the absorbance scale with the right-hand knob. The reagent blank is a solution that contains all the chemicals that are in the other samples except the molecule of interest. 5. Transfer your palest sample to the cuvette, insert it into the sample compartment, and read the absorbance value and record it in Table 3. 6. Pour the sample back into the test tube it came from and continue reading the absorbance of the other samples from palest to darkest (don t discard any samples until you are finished and are sure that your readings are accurate).
Biol3460 Lab 1 4 Table 3. Absorbance values for solutions of known BSA concentration, measured by spectrophotometer at 595 nm. BSA concentration (mg/ml) Absorbance (A 595 ) 1.0 0.75 0.50 0.25 0.10 0.05 0.0 Part E. Standard Curves and Graphing Formats Proper graph construction is a must in nearly every course of biological study. Graphs provide a clear indication of general trends seen in a range of data and can be used to infer relationships between variables. A standard curve is a graph in which the absorbance of a sample is plotted as a function of various concentrations of the compound of interest. The equation of this curve can then be used to determine the concentration of unknown samples of the same compound, when you have measured their absorbance. Standard Curve Construction: 1. Plot the absorbance values obtained from Part D against the known concentrations of the seven protein solutions. 2. Be sure to properly label the axis and provide a descriptive figure caption for the graph. 3. Fit a straight line to the data and calculate the equation of the line. 4. Use the equation of the line to determine the concentration of the two unknown samples you ran with the protein assay in Part D. Graph Formatting Tips: Graphs should be constructed in Excel or a similar spreadsheet program, however you may use graph paper if you are very neat. The dependent variable is always plotted on the y-axis against the independent variable, which is plotted on the x-axis. The axes scales should be divided into equal intervals if the data is continuous. Choose intervals that are logically spaced and permit easy interpretation of the graph. If there are no data points at the low end of the scale it is not necessary to start the scale at zero. However, do not break the graph in the middle of the scale with data points on either side. Axes labels should be descriptive and should include the units for the measurement in parentheses. The graph should include a descriptive figure caption, placed below the graph. Label the graph as a figure and give it a number (e.g. Figure 1), based on when it is referred to in the text of your paper. The description should include all information necessary to understand the data presented in the graph (all variables, treatments, types of values reported, symbols, sample sizes, and organisms or cells studied). To illustrate trends in line graphs, use smooth curves or straight lines to fit the values for one data set. Do not force a straight line through a data set that does not appear to be linear and do not force a straight line through the origin (0,0) if it is not representative of the data series.
Biol3460 Lab 1 5 Conversion Factors Table 4. The calibrated values of an ocular unit for various light microscope objective lenses as determined by a stage micrometer. Objective Lens Single Ocular Unit Value (mm) 4X 0.026 10X 0.01 40X 0.0026 100X 0.001 Metric Conversion Values: Volume 1L = 1000 ml 1 ml = 1000 µl Length 1 m = 100 cm 1 cm = 10 mm 1 mm = 1000 µm 1 µm = 1000 nm Mass 1 kg = 1000 g 1 g = 1000 mg 1 mg = 1000 µg