Life at Ryerson is the study of Biology in the Department of Chemistry and Biology -­‐ Gilbride, 2011 Biology is the study of life How do we do that? A. Observing Life
The function of any microscope is to magnify objects without losing resolution. Magnification is the ability of the microscope to create an enlarged view of an object such that we can observe details not otherwise possible with the human eye. Resolution is the ability to distinguish between two distinct images. If we magnify too much without increasing the resolution then the objects will be very large but also very blurry and it will be hard to see if two objects very close together are actually two. Figure 1.1 Magnification vs. Resolution
If an image of a cell is magnified from 10X to 45X, the image gets larger, but not necessarily any clearer. The image on the left in Figure 1.1 is magnified with no increase in resolution. The image on the right is magnified the same, but with increasing resolution. Note that by the time the image is magnified 10X (from 10X to 100X), the image on the left is completely unusable. The image on the right, however, presents more detailed information. Without resolution, no matter how much the image is magnified, the amount of observable detail is fixed, and regardless of how much you increase the size of the image, no more detail can be seen. At this point, you will have reached the limit of resolution or the resolving power of the lens. Figure 1.2 shows all the parts of the microscope that will be used in the lab activities. Table 1.1 lists the functions of these parts. 1 Figure 1.2 Front and side view of the Leica CME Binocular Microscope.
Table 1.1 Microscope Components and Their Functions Functions Microscope Component Ocular (eye piece) Magnification (10x) Body tube Holds oculars, conducts light rays from specimen Nose piece Holds objectives Low-­‐power objective (10x) Magnification, used for scanning specimen High-­‐dry power objective (40x) Magnification Oil immersion objective (100x) Magnification, used for bacteria Condenser Concentrates and directs light beam through specimen Stage Specimen support and movement Mechanical stage Manipulation of specimen’s location Condenser (iris) diaphragm Regulates amount of light passing through lenses Coarse adjustment knob (focus) Used for preliminary and coarse focusing by raising or lowering stage or body tube Fine adjustment knob (specimen focus) Used for final or fine focusing by raising or lowering stage or body tube Condenser adjustment knob Used to obtain full illumination by raising or lowering condenser Light Intensity knob Adjust brightness of light Base and Stand (sometimes called arm) Microscope support 2 Exercise 1: Proper use of the Microscope Activity 1: Understanding parts and function of the microscope 1. Observe the microscope you will be using and identify parts of microscope and learn its functions. 2. Look through the eyepiece and ensure that the lenses are clean. Use lens paper and lens cleaner to wipe dirt on any of the lenses. The lab instructor will guide you on what to look for when checking which lens is dirty. 3. Once your microscope is cleaned, it is ready for use. Activity 2: Understanding depth and focus 1. Place a slide containing three colored and crossed threads on the microscope stage. 2. Locate a spot where all three threads cross each other at the same point. Use the coarse adjustment knob to first focus on the threads until you see a clear image. Use the fine adjustment knob control to focus first on the lowermost thread, then the middle thread and finally, the uppermost thread. List the order of the threads from the top to the bottom, by indicating their color on Table 1.2. Table 1.2 Order of the Threads in the Prepared Slides Position Colour Top Middle Bottom Activity 3: Preparing a wet mount slide and observing a pond organism. 1. Make a wet mount slide of Daphnia magna (water flea) on a depression slide by pipetting the organism from the beaker and placing a drop of sample onto the well of the depression slide. 2. Place the slide on the stage of the microscope. Use the low power lens (4X objective) to look at the organism. Can you see all the parts shown in Fig. 1.3 of this animal? Does the microscope enable you to view the organism better than you own eyes? Figure 1.3 Image and diagram of Daphnia. 3 B. Experimenting with Life
Some molecules, like proteins and sugars, are able to dissolve in water. Sometimes these molecules will ionize, that is, separate into charged fragments. For example, table salt (NaCl) when dissolved in water will separate into Na+ and Cl-­‐. These ions (charged fragments) can be either anions or cations. Anions are charged molecules with an overall negative charge, for example the Cl-­‐ ion. Cations are charged molecules with an overall positive charge, for example the Na+ ion. The water molecule (H2O) can ionize into H+ and OH-­‐. Pure water has an equal concentration of H+ and OH-­‐ ions and measures a pH of 7. Some solutions can have higher or lesser amounts of each of these ions. A solution that has more H+ ions than OH-­‐ ions is called an acidic solution (pH < 7). A solution that has more OH-­‐ ions than H+ ions is called a basic/alkaline solution (pH > 7). When acids and bases are mixed together, a neutral solution can be created. The pH scale (Table 1.4) expresses the concentration of hydrogen ions from 0 (acidic) to 14 (alkaline). There is a tenfold difference in hydrogen ion concentration between each pH unit. There are different methods, such as a pH meter and color indicators, that can be used to determine the pH of a solution. Color indicators are usually chemical solutions that will change color as the pH of the solution changes. pH paper is paper that is saturated with indicator that you can dip into a solution to test the pH. pH 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Table 1.4: The pH Scale and Common Examples of Acids and Bases [H ] (molar) [OH-­‐] (molar) Relative Strength Examples +
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10 Strong Acid Moderate Acid Weak Acid Neutral Weak Base Moderate Base Strong Base Hydrochloric acid Pure Water Sodium Hydroxide Exercise 2: Using Red Cabbage Indicator vs. pH Meter to Measure pH Red cabbage has its colour due to the pigment, anthocyanin. Anthocyanin is responsible for the red, blue, and purple colours found in plants and vegetables. These pigments will change colour as pH changes. The red cabbage has been boiled before the lab in order to extract the anthocyanin pigments. Activity 4: Preparation of Colour pH Standard a) b) b) c) Label 7 clean test tubes with pH 2, 4, 6, 7, 8, 10 and 12. Add 20 drops of red cabbage extract into each of clean labelled test tube Put one dropper full of the pH buffer solution into corresponding labeled test tubes. Fill in results in Table 1.5. 4 Table 1.5: Colorimetric pH Standard using Red Cabbage Extract as pH Indicator Colour Acid or Base pH 2 4 6 7 8 10 *12-­‐ initial *12-­‐final * The pigments are not stable at this pH 12, so record both final and initial colours. Activity 5: pH Analysis of Drinks and Common Stomach Medications 1.
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Label 8 test tubes with samples used in Table 1.6. Add 10 drops full of red cabbage extract into each tube. Put 1 dropper full of the each sample into corresponding labeled test tubes. Compare the resulting color to your set of pH standards created earlier. Record your results in Table 1.6. 5. Using a pH meter, take a pH reading of each of the samples and record your results in Table 1.5 Table 1.6: pH Values of Beverages and Medications Beverages pH using colour standard pH using pH meter Medicines White grape juice Milk of Magnesia (Mg(OH)2) 7-­‐UP Sodium Bicarbonate (NaHCO3) White Wine Maalox Seltzer Water Lemonade pH using colour standard pH using pH meter Questions: 1. What type of solutions are most drinks? 2. What type of solutions are most stomach medications? Why do you think this is so? 3. How do the pH measurements using the red cabbage extract compare with the pH measurements using the pH meter? 5 Exercise 3: Fermentation Of Carbohydrates By Yeast Organisms can extract chemical energy from carbohydrates in the absence of molecular oxygen by fermentation. The end products are either lactic acid or alcohol. During anaerobic glycolysis, one molecule of glucose is converted to two molecules of lactic acid while in alcohol fermentation; one molecule of glucose produces two molecules of ethanol and two molecules of carbon dioxide. In the absence of oxygen, yeast obtain chemical energy from glucose and other sugars by alcoholic fermentation. During this experiment, the speed at which a given sugar is metabolized is observed directly by measuring the amount of carbon dioxide produced. Water is displaced from an inverted test tube by the carbon dioxide. The length of the column of gas is directly proportional to the rate of alcoholic fermentation in the presence of a given sugar. The differences in the amount of carbon dioxide produced can be accounted for by the presence or absence of the enzymes required for the catabolism of the molecule, the length of the catabolic pathway and/or the rate of the enzyme catalyzed reactions in the pathway. Activity 6: Determining Rates of Fermentation for Various Sugar Solutions 1.
Place a narrow ring of masking tape around each of seven test tubes near their closed ends. The ends of the tape should be overlapped to form a tab. 2.
Number the tabs 1 to 7 3.
Pipette 0.5mL of a 15% yeast suspension into each tube. 4.
Fill each test tube with one of the 10% sugar solutions provided. 5.
Plug each test tube with a cotton ball that has been soaked in the corresponding sugar solution. 6.
Invert the tube in a warm water-­‐filled beaker (250mL) ensuring that no air enters the test tube. 7.
Place the 250mL beaker in a 600mL beaker and fill the large beaker with warm water until the test tubes are nearly submerged. 8.
Periodically check to make sure this water is still warm. 9.
Cover the 600mL beaker with a watch-­‐glass to prevent the test tubes from falling over as they fill with gas. 10.
Maintain an elevated temperature until one of the tubes has filled with gas. 11.
Measure the length of the gas column in each tube after first raising it in the beaker of water to equalize the pressure in the tube with that of the atmosphere. DO NOT REMOVE the tube completely from the beaker until you have a measurement. 6 Table 1.6: Amounts of gas produced by fermenting yeast Sugar Length of Gas Column (mm) Control Fructose Glucose Lactose Maltose Ribose Starch Sucrose Questions: 1. List the lengths of the gas columns in descending order (Longest to shortest). 2. Did the yeast possess the enzyme(s) required for the catabolism of all the carbohydrates that were used in this experiment? If not which carbohydrates were not catabolized? Explain why a given carbohydrate(s) is not catabolized. 3. Explain the differences in the length of the gas column you obtained for the carbohydrates which were catabolized. 7