The University of Lethbridge

BIOLOGY 1010 LABORATORY MANUAL Spring, 2008

BIOLOGY 1010 The Cellular Basis of Life Laboratory Manual

Spring, 2008 Written by: Helena Danyk Department of Biological Sciences University of Lethbridge

Student Name: Lab Day:

I.D. Number: Lab Section:

Lab Time:

Instructor’s Name:

Office:

Email address:

Telephone:

Office Hours:

Lab Room:

TABLE OF CONTENTS Exercise

Page #

Spring 2008 Laboratory Schedule………….…………….………………………….…….i Evaluation and Grading Policies ………………………………………………….……...ii Safety Procedures ………………………………...………………………………......….iii Cell Structure and Function, Part 1 …………………………………………………….....1 Cell Structure and Function, Part 2 …………………………………………………...…..8 Bacteriology, Part 1 ……………………………………………………………..………17 Bacteriology, Part 2 ……………………………………………………………….…….28 Cell Membranes: Effects of Stress ……………………………………………...……….36 Enzymes …………………………………………………………………………………46 Fermentation and the Scientific Method ……………………………………………..….55 DNA Structure and Function……………………………………………………….……63 Appendix A: How to Use the Microscope ………………………………………………72 Appendix B: How to Make a Scientific Drawing ……………………………………….75 Appendix C: How to Determine the Size of a Specimen ………………………………..77 Appendix D: Aseptic Technique ……………………………………………………..….78 Appendix E: How to Use a Spectrophotometer ……………………………………....…79 Appendix F: Scientific Inquiry ………………………………………………………….82 Appendix G: Conversions and Taxonomy …………………………………………..…..91 REVISED: Summer, 2007 Written By Helena C. Danyk University of Lethbridge, Lethbridge, AB ©2007-08

i

BIOLOGY 1010 LABORATORY SCHEDULE: Spring, 2008 Jan. 7-11

Cell Structure and Function, Part 1

Jan. 14-18

Cell Structure and Function, Part 2

Jan. 21-25

Bacteriology, Part 1

Jan. 28-Feb. 1

Bacteriology, Part 2

Feb. 4-8

Bacteriology, Part 2 – Complete; Scientific Method

Feb. 11-15

Cell Membranes: Effects of Stress

Feb. 18-22

Reading Week – no labs this week

Feb. 25-29

Enzymes

Mar. 3-7

Fermentation, Part 1

Mar. 10-14

Fermentation, Part 2

Mar. 17-21

Good Friday – no labs this week

Mar. 24-28

Easter Monday – no labs this week

Mar. 31-Apr. 4

DNA Structure and Function

Monday April 7

Final Lab Exam –4:00-6:00 p.m., or 6:30-8:30 p.m.; in PE 250

For help or advice on any aspect of the Biology 1010 laboratory, please consult your lab instructor or the lab coordinator (Helena Danyk: D884, 329-2664, [email protected]).

ii Evaluation: The laboratory portion of Biology 1010 is worth 35% of the overall course grade, and is calculated based on the following allocations: In-class Quizzes and Assignments: Laboratory Report: Final Lab Exam:

14.5% 8% 12.5%

Attendance: You may only attend the lab section in which you are registered due to safety regulations, equipment limitations, and room capacities. However, if you are aware of a planned absence, consult your lab instructor who will give you permission to attend an alternate section if space is available. If you miss a lab due to illness or personal emergency, inform your instructor as soon as possible so that alternate lab arrangements can be made. Your lab instructor may request documentation from you (doctor’s note, coach’s letter, etc) at his/her discretion. It is your responsibility to ensure that you are properly prepared for all lab quizzes, assignments and exams even if you are not in the lab. Labs are given one week at a time and are not repeated in subsequent weeks, so if you miss an entire week of classes you will not be able to make up the lab exercise. Missed Assignment and Exam Policy: In-class quizzes and assignments must be written as scheduled, except in the case of a medical excuse or personal emergency (validity of excuse to be determined on a case-bycase basis by your instructor, in the event that documentation cannot be provided). If you must be absent for an approved reason, you will be excused from the quiz or assignment and the weight of the missed work will be adjusted for in your final lab mark. There are NO make-up quizzes or assignments. Students will be required to write and submit a lab report regardless of whether they attended the relevant lab or not. If you miss the final lab exam for any reason other than medical as previously outlined, you will be required to apply for an incomplete in the course and write the final lab exam in the following semester.

iii GUIDELINES FOR SAFETY PROCEDURES Students enrolled in laboratories in the Biological Sciences should be aware that there are risks of personal injury through accidents (fire, explosion, exposure to biohazardous materials, corrosive chemicals, fumes, cuts, etc). The guidelines outlined below are designed to: a) minimize the risk of injury by emphasizing safety precautions and b) clarify emergency procedures should an accident occur. EMERGENCY NUMBERS:

City Emergency Campus Emergency Campus Security Student Health Centre

911 2345 2603 2484 (Emergency - 2483)

THE LABORATORY INSTRUCTOR MUST BE NOTIFIED AS SOON AS POSSIBLE AFTER THE INCIDENT OCCURS.

EMERGENCY EQUIPMENT:

Your lab instructor will indicate the location of the following items to you at the beginning of the first lab period. 1. 2. 3. 4. 5. 6. 7.

Closest emergency exit Closest emergency telephone and emergency phone numbers Closest fire alarm Fire extinguisher and explanation of use Safety showers and explanation of operation Eyewash facilities and explanation of operation First aid kit

GENERAL SAFETY REGULATIONS:

• •

• • • • •

Eating and drinking is prohibited in the laboratory. Keep pencils, fingers and other objects away from your mouth. These measures are to ensure your safety and prevent accidental ingestion of chemicals or microorganisms. Coats, knapsacks, briefcases, etc. are to be hung on the hooks provided, stowed in the cupboards beneath the countertops, or placed along a side designated by your instructor. Take only the absolute essentials needed to complete the exercise* with you to your laboratory bench. (* e.g. manual, pen or pencil) Mouth pipetting is NOT permitted; pipet pumps are provided and must be used. Always wash your hands prior to leaving the laboratory. Students are not allowed access to the central Biology Stores area for any reason. Consult your instructor if you require additional supplies. Report any equipment problems to instructor immediately. Do NOT attempt to fix any of the equipment that malfunctions during the course of the lab. Use caution when handling chemical solutions. Consult the lab instructor for instruction regarding the clean-up of corrosive or toxic chemicals.

iv Contain and wipe up any spills immediately and notify your lab instructor (see SPILLS below). Heed any special instructions outlined in the lab manual, those given by the instructor or those written on reagent bottles. Long hair must be restrained to prevent it from being caught in equipment, Bunsen burners, chemicals, etc. Dispose of broken glass, microscope slides, coverslips and pipets in the specially marked white and blue boxes. There will be NO disposal of glassware in the wastepaper baskets. You are responsible for leaving your lab bench clean and tidy. Glassware must be thoroughly rinsed and placed on paper toweling to dry.

• • • •

SPILLS:

•

Spill of SOLUTION/CHEMICAL: While wearing gloves, wipe up the spill using paper towels and a sponge as indicated by the lab instructor.

•

Spill of ACID/BASE/TOXIN: Contact instructor immediately. DO NOT TOUCH.

•

BACTERIA SPILLS: If necessary, remove any contaminated clothing. Prevent anyone from going near the spill. Cover the spill with 10% bleach and leave for 10 minutes before wiping up. Discard paper towels in biohazard bag. Discard contaminated broken glass in designated biohazard sharps container.

DISPOSAL:

•

Broken glass, Pasteur pipets, pipets are placed in the upright white ‘broken glass’ cardboard boxes. NO PAPER, CHEMICAL, BIOLOGICAL OR BACTERIAL WASTE MATERIALS should be placed in this container

•

Petri plates, microfuge tubes, pipet tips should be placed in the orange biohazard bags. The material in this bag will be autoclaved prior to disposal.

•

Bacterial cultures in tubes or flasks should be placed in marked trays for autoclaving.

•

Liquid chemicals should be disposed of as indicated by the instructor. DO NOT dispose of residual solution in the regent bottles. In case of any uncertainty in disposal please consult the lab instructor.

•

Slides of bacteria should be placed in the trays filled with 10% bleach that are located at the ends of the laboratory benches.

HEALTH CONCERNS:

Students who have allergies, are pregnant, or who may have other health concerns should inform their lab instructor so that appropriate precautions may be taken where necessary.

v THE UNIVERSITY OF LETHBRIDGE Policies and Procedures Occupational Health and Safety Manual

SUBJECT:

CHEMICAL SPILLS PROCEDURE

Precaution should be taken when approaching any chemical spill. 1.

UNKNOWN SPILL a. Clear the area b. Call Security at 329-2345 c. Secure the area and do not let anyone enter d. Call Utilities at 329-2600 and request air be turned on at the spill site e. Security will respond and determine the severity of the spill f. Security will immediately notify the spill team as follows: • Peter Dibble 331-5201 • Michael Gerken 332-2173 • DBS Environmental only if above not available 328-4483 (24 hrs) • U of L Occupational Health and Safety 332-2350 (Carolin) 394-8716 (Bill)

2.

KNOWN SPILL a. Clear the area b. Call Security at 329-2345 c. Secure the area d. Call Utilities at 329-2600 and request air be turned on at the spill site e. Security will respond and determine the severity of the spill f. Security will immediately notify the spill team as follows: • Peter Dibble 331-5201 • Michael Gerken 332-2173 • DBS Environmental only if above not available 328-4483 (24 hrs) • U of L Occupational Health and Safety 332-2350 (Carolin) 394-8716 (Bill)

3.

NOTIFICATION a. Occupational Health and Safety will notify the appropriate departments, including notification of appropriate government agency.

h:\shared\security\Chemical Spills Procedure.doc

1

CELL STRUCTURE AND FUNCTION: PART I Introduction: Cells are considered the basic unit of all living organisms because they perform all of the processes we call ‘life’. All organisms are composed of cells. Although most individual cells are visible only with the aid of a microscope, some may be up to a meter long (e.g. nerve cells) or as large as a small orange (e.g. the yolk of an ostrich egg). Despite such differences, all cells have a similar fundamental design and share many features. The objectives of today’s lab are to: • examine some of the features of cells as a means of understanding the life processes of organisms • become familiar with the structures and organelles possessed by cells, and to determine the relative sizes and functions of each • provide an introduction to microscopy, including the use and care of the microscope and micrometry to measure cell and organelle size • learn how to present information in scientific drawings As background for this lab and the following exercises, you will find it useful to read pages 6-8 and 94-122 in Campbell and Reece (2005).

A. Elodea Cells Elodea is a widely distributed pond weed that is often used in studies of photosynthesis. Elodea is a eukaryote, meaning its cells contain membrane-bound nuclei and other organelles. For example, chloroplasts are elliptical green structures (organelles) in the cells. Chloroplasts are the site of photosynthesis and are green because of the presence of the photosynthetic pigment chlorophyll. Chloroplasts are a feature of plant cells. Remove a young leaf from the tip of a sprig of Elodea. Place this leaf in a drop of water on a microscope slide and cover it with a coverslip. Do not let the leaf desiccate (dry up). Examine the leaf with the 10x power and then the 40x power objective lens. Each of the regularly shaped units you see is a cell, and each is delimited by a cell wall made of cellulose. Cellulose is a complex carbohydrate formed of glucose molecules attached end to end. Cell walls are another characteristic of plant cells. Most plant cells contain a large central vacuole surrounded by a membrane called the tonoplast. As a consequence, the liquid cytoplasm containing the chloroplasts and other organelles is restricted to the periphery of the cell. Look carefully at the periphery of the cells and chances are you will detect a slow, circular movement of the chloroplasts; this phenomenon is called cytoplasmic streaming. Because the central vacuole of Elodea does not contain coloured material, it is sometimes difficult to discern. Chloroplasts that appear to be in the center of the cell are actually around the edges. To confirm this, focus up and down through the cell. The vacuole occupies approximately 90% of the volume of each Elodea cell

2 and contains water and a variety of dissolved materials such as sugars, alkaloids and inorganic salts. In general, vacuoles serve as storage areas for food materials and depots for waste materials. Vacuoles also help maintain cell turgidity and thus cell shape. Draw representative Elodea cells in the space below and label them. Refer to Appendix B for rules regarding scientific drawings. Use an ocular micrometer (Appendix C) to measure the lengths and widths of five cells. Convert ocular units to millimeters, and calculate means (Appendix F) for length and width. Use the mean length value to calculate your drawing magnification. As well, measure and report the average diameter of a chloroplast.

B. Structure of Plastids Plastids are organelles of plants that are the sites of such activities as food manufacture and storage. You have already examined chloroplasts, a type of plastid in which photosynthesis occurs. However, other plastids have different functions. You will examine two other kinds of plastids – amyloplasts and chromoplasts. Use a razor blade to make a thin section of potato tuber (make this section as thin as possible). Stain the section for a few seconds with iodine, a stain specific for starch. Add a coverslip, and examine under the microscope. The intensely stained structures in the cells are amyloplasts, a type of plastid that stores starch. Draw a potato cell in the space below. Calculate drawing magnification and include this information in your figure caption.

3 •

Measure the smallest and largest amyloplasts you can find and report the size range.

Use a razor blade to prepare a thin section of red pepper. Place the section in a drop of water on a glass slide, add a coverslip, and examine with your microscope. The tiny pinpoint orange organelles are chromoplasts, a type of plastid containing pigments other than chlorophyll (in this case, probably carotenes and/or xanthophylls). Draw red pepper cells in the space below.

•

Compare the size of the chloroplasts, amyloplasts and chromoplasts you have observed thus far.

C. Paramecium Add a drop of Paramecia culture to a tiny drop of “Protoslo” (methyl cellulose) in order to slow down the movement of the Paramecia. Mix gently with a toothpick and add a coverslip. Observe your slide with the 4x, then the 10x objective lens to locate a Paramecium. If it is not moving too quickly, you should be able to switch over to the 40x objective lens to observe fine details of its structure. Note the beating of the cilia. This serves two purposes; it propels the organism and it sweeps food into an oral groove. As the food moves down to the bottom of the groove it is taken into a food vacuole and digested as the vacuole moves around the cell. You should be able to see the contractile vacuole, a perfectly round and clear vesicle. It slowly builds in size, then fuses with the plasma membrane and contracts to expel its contents (water) from the cell. Use the space below to sketch your Paramecium and label the parts you observed. What is the size of a Paramecium?

4

D. Human Epithelial Cells Gently scrape the inside of your cheek with the broad end of a toothpick. Stir the scrapings into a drop of methylene blue on a glass slide, add a coverslip, and observe under the 10x power lens, then the 40x lens of the microscope. Use the space below to prepare a scientific drawing.

•

You may have noticed that some of the cells may have had their edges folded over. What does this indicate about the thickness of the cells?

E. Pond Water If available, prepare a wet mount of water from the aquarium provided. View your slide under the low power objective lens initially. You should see examples of autotrophic (photosynthetic) organisms as well as heterotrophic (ingestive, animal-like organisms). Consult reference materials available to see if you can identify any of the organisms, and make some representative sketches in the space below.

5

F. Unusual Cell Types View the demonstration of unusual cell types present at the back of the lab. Each of these represents a single cell. Can you think of any other unusual cell types not shown here? Do you have any thoughts as to why there is such variation in size and shape of particular cells?

6

Thought Questions: 1. Think of the cells you viewed today in lab. Try to correlate cell structure to function. If a cell has an abundance of certain organelles, can you infer the major function of the cell?

2. Why are most cells microscopic (refer to Figure 6.7 of Campbell and Reece, p. 99)? In this context, is it better for a plant to have several large chloroplasts or many small chloroplasts? Explain your answer.

3. What is the usefulness of cytoplasmic streaming?

4. Students often confuse the cell wall with the cell membrane. Compare the structure and function of the cell wall and cell membrane. Do all cells have both?

5. An inherited disorder in humans results in the absence of a certain critical protein in flagella and cilia. The disease causes respiratory problems and in males, sterility. What is the ultrastructural connection between these two symptoms?

6. Why are contractile vacuoles necessary in Paramecium?

7 7. Complete the following Table: Structure

Function

Cell Wall Nucleus Vacuole Chloroplast Mitochondrion Contractile Vacuole

8. Human epithelial cells are highly specialized, for protection. As a consequence of specialization, they have lost the ability to carry out certain cellular functions. Describe some of the functions that epithelial cells may have lost.

Literature Cited Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San Francisco, CA.

8

CELL STRUCTURE AND FUNCTION: PART II Introduction: In your laboratory last week, you examined a diversity of eukaryotic cell types, including animal, plant and protist cells, single-celled organisms capable of performing all of the functions associated with life, and cells from multicellular organisms that were specialized to perform a limited number of tasks. You were also introduced to some of the structures and organelles associated with eukaryotic cells. You will continue your study of cell structure and function in this exercise. Today’s laboratory will focus more specifically on processes used by cell biologists to study particular cell constituents, namely plasma membranes, chloroplasts and mitochondria. In the first part of the exercise, we will use artificial membranes to study the selective permeability of membranes, and then will look at the same process in red onion cells. Part two of today’s lab will make use of centrifugation to isolate cell constituents. A suspension of cell parts and organelles (termed a homogenate) may be separated using differential centrifugation. Differential centrifugation relies on using different amounts of centrifugal force to sediment cell parts of different densities and sizes, and is a technique commonly used by cell biologists to study cells. The objectives of today’s lab are to: • learn about the semi-permeable nature of the plasma membrane • investigate the roles played by osmosis and diffusion in the proper functioning of a cell • make use of differential centrifugation techniques with the purpose of separating and identifying cell components Please read pages 97, 109-111, and 130-133 in Campbell and Reece (2005) in preparation for this exercise.

A. Diffusion Through a Selectively Permeable Membrane One of the most important properties of a cell membrane is its ability to regulate the passage across of ions and molecules. Typically carbon dioxide and oxygen can pass through membranes easily, while larger, hydrophilic molecules (those possessing a net charge) like sugars are impeded. The movement across membranes of molecules like these is facilitated by the presence of transport proteins imbedded in the membrane. We will examine the structure of cell membranes in more detail in a future lab. Today we will concentrate on some of the mechanisms that drive substances across membranes, namely diffusion and osmosis. Diffusion, the movement of a substance from an area where it is more concentrated to an area where it is less concentrated, is a spontaneous process that requires no input of energy (passive transport). This process explains much of the movement of molecules across cell membranes. The diffusion of water across a selectively permeable membrane is a special kind of passive transport termed osmosis. The direction that water moves depends on the

9 total amount of solute present on each side of the membrane. A solution with a higher concentration of solutes compared to another is referred to as a hypertonic solution, while the solution with the lower concentration of solutes is termed a hypotonic solution. If the net concentrations of solutes are equal in both solutions, they are said to be isotonic. Water always moves from a hypotonic to a hypertonic solution. We can make use of a simple model system to illustrate both of these concepts. Cellophane dialysis tubing acts as a synthetic semi-permeable membrane that permits the diffusion of water and other small molecules while prohibiting the movement of larger molecules. Work in pairs to create and manipulate artificial cells using the procedures outlined below. Pair 1: • Cut a 10 cm strip of tubing, and immerse in the container of water provided for about a minute, until the tubing can be opened easily by rolling between your thumb and fingers. • Tie a knot near one end of the tubing using the string provided. Use a plastic disposable pipette to fill the tube to within 5 cm of the top with an 80% glucose solution. • Insert a plastic 1 mL pipette into the tubing, and securely tie the open end of the tubing shut around the pipette. • Use the stands provided to support the bag in a beaker of water. • Observe the position of the column of fluid in the pipette periodically for about 30 minutes. If there is no change after the first five minutes, check your set-up for leaks (which will appear as wiggly lines in the beaker of water). Record your observations below.

Pair 2: • Cut a 10 cm strip of tubing, and immerse in the container of water provided for about a minute, until the tubing can be opened easily by rolling between your thumb and fingers. • Tie a knot near one end of the tubing using the string provided; then use a Pasteur pipette to fill the tubing with starch solution. Tie the other end of the tubing. • Immerse the tubing in a beaker of water to which you have added enough iodine to make the solution light brown in colour. • When starch molecules come into contact with iodine, a blue or purplish colour appears (what evidence do we have for this from last week’s lab?). Observe the bag and the solution for about 15 minutes and record any colour changes that you observe.

10 Can iodine pass through the membrane? How do you know?

Can starch pass through the membrane? How do you know?

Which experiment demonstrates osmosis?

Why is the other experiment not directly a demonstration of osmosis?

Osmosis can also be easily observed in living cells. Work individually to complete the next part of the exercise: •

Take one of the red onion leaves provided for you on the side bench and snap the leaf backwards. Peel back the thin piece of red epidermis formed at the break point (your instructor will demonstrate this). Place this epidermal tissue in a drop of water on a microscope slide, add a coverslip, and examine. The entire cell appears red due to pigment granules (anthocyanin) dissolved within the central vacuole. Sketch what you see in the space below.

•

Prepare another wet mount as you did above, but replace the water on the microscope slide with 2-3 drops of 20% NaCl. Add a coverslip, examine, and sketch your observations in the space below. How do the cells differ in appearance from those viewed above?

11 The shrinkage of the cytoplasm because of osmotic water loss is called plasmolysis (Figure 1).

Figure 1: Plasmolysis of a Plant Cell In the first preparation, which solution is hypertonic, the water the cell is immersed in, or the water inside the cell?

In the second preparation, which solution is hypotonic, the NaCl solution the cell is immersed in, or the water inside the cell?

B. Cell Fractionation Cell fractionation is a process in which cells are gently broken apart and their components separated. Cellular organelles remain intact and biochemically active so that their functions may be studied. Cells can be disrupted by a variety of means, including electric shock, sonication (vibration) or grinding. The resulting suspension of cell parts and organelles, called a homogenate, can then be separated using differential centrifugation. At low speeds, large nuclei and intact cells sediment to form a pellet at the bottom of the centrifuge tube, while the remaining organelles are found in the supernatant above the pellet. Centrifugation of the supernatant at successively higher speeds isolates successively smaller components of the cell in pellets. We will make use of differential centrifugation to isolate chloroplasts and mitochondria from a combined pea and spinach homogenate. We will then examine the different fractions resulting from our centrifugation procedure for the presence or absence of these two types of organelles. Please work in groups of four to complete the following experiment. Each group of four does the following: 1. Fill two 15 mL centrifuge tubes to the 12 mL mark with the pea-spinach homogenate located at the side bench (the homogenate was prepared by your instructor just prior to the lab period by placing pea seeds soaked overnight together with two or three

12 spinach leaves in a blender with phosphate buffer, blending briefly, and then straining through cheesecloth). Label the tubes with your name and bench number. 2. Place your tubes in the centrifuge (the rotor must always be balanced with equal weights or volumes opposite each other). Centrifuge at 200x gravity for three minutes. 3. While your material is in the centrifuge, remove a small sample of the cellular debris from the cheesecloth and prepare a wet mount. Place a coverslip on the slide, and observe the material using the low power objective. •

Do you see cell wall fragments?

•

Do you see intact cells?

•

Prepare another slide, but use iodine solution in place of water. What do you see? Sketch your observations in the space below.

•

Use the ocular micrometers provided to measure the length of 10 of the stained structures. Use these values to calculate their mean size, referring back to Appendix C if necessary. Record your measurements in Table 1.

4. After centrifugation is complete, remove your test tubes from the centrifuge. Carefully decant the supernatants into two new centrifuge tubes. Store the original tubes (each containing a white pellet) on ice for the time being. Return the new tubes to the centrifuge and centrifuge at 1300xg for 10 minutes. 5. Meanwhile, examine the white pellet collected in step 4. Mix a drop of iodine with a small amount of the pellet on a microscope slide, add a coverslip, and observe (pea seedlings store their food reserves in the form of starch grains). • Describe the appearance of the starch grains you observe.

13 •

Use the micrometer to measure the size of 10 of these starch grains. Has their mean size changed? Record your measurements in Table 1. 6. After the second centrifugation is complete, carefully remove your test tubes from the centrifuge. You should see a green layer above another small white pellet. This green layer will contain the nuclei and chloroplasts. 7. Use a clean Pasteur pipette to carefully remove about 2 pipettes full of the yellowishgreen supernatant. Place it into a new test tube, and store the tube on ice until needed (the supernatant contains the mitochondrial fraction). The remainder of the supernatant from your two tubes can be carefully pipeted into a waste beaker. Once the supernatant has been removed, carefully remove a drop of the green layer from one of your tubes and place it onto a microscope slide. Add a drop of water and a coverslip, and examine it with your microscope using the low power, and subsequently, the high power objective lenses. •

Do you see aggregations of green-coloured bodies? What are these?

•

Calculate the mean length of 10 of the green structures observed, and record your values in Table 1.

•

In some cases nuclei may also be visible. They will appear as large, round, grayish structures.

8. Make another wet mount of the green residue, but this time use iodine in place of water. •

Are amyloplasts still visible? If so, compare the size of these amyloplasts with those isolated in steps 3 and 5. Why might the sizes be different?

9. Prepare a wet mount of the yellowish-green supernatant from step 7. Add a drop of Janus Green stain to this wet mount. You should see clumps of small, blue-stained objects; these are the mitochondria. •

Use the micrometer to measure and record the size of 10 of the mitochondria you observe; record values obtained in Table 1.

•

How does mitochondrial size differ compared to chloroplast size?

14

Table 1: Group data for measurements of cells and organelles at various stages in the fractionation procedure. Cell Fraction:

Cell/Organelle Size Measurements (mm)

Mean Size (mm)

Cheesecloth Debris -intact cells -amyloplasts Pellet #1 -amyloplasts Green Layer -chloroplasts -amyloplasts Yellow Supernatant -mitochondria

Table 2: Class data for measurements of cells and organelles at various stages in the fractionation procedure. Cell Fraction: Cheesecloth Debris -intact cells -amyloplasts Pellet #1 -amyloplasts Green Layer -chloroplasts -amyloplasts Yellow Supernatant -mitochondria

Cell/Organelle Size Measurements (mm)

Mean Size (mm)

15

16

Thought Questions 1a). What would happen if the Paramecium cells you viewed last week were placed in a solution of pure water? Use the terms osmosis, hypertonic and hypotonic in your answer.

1b). What role does the contractile vacuole play in maintaining osmotic equilibrium in a Paramecium’s natural environment?

1c). Would the same be true of Elodea cells placed in pure water? Why or why not?

2.

What physical properties of cell organelles determine their behaviour during differential centrifugation? Why are different speeds of centrifugation used? Rank the cell parts that you have studied over the last two periods based on their size (smallest to largest).

17 3. Organelles are often studied by separating them on a sucrose gradient. A centrifuge tube is filled with a sucrose solution of increasing concentration from the top of the tube to the bottom. What would you expect to observe if you put a layer of cell homogenate on top of the sucrose and then subjected the sample to high speed centrifugation?

4. Janus Green is blue when in an oxidized state, and as it is reduced, it becomes colourless. Why was Janus Green a useful stain to use to identify mitochondria in this exercise? What would you expect to happen if you had examined your slide again after several hours? Why?

Literature Cited: Helms, D.R., Helms, C.W., Kosinski, R.J. and Cummings, J.R. 1998. Biology in the Laboratory 3rd Ed. W.H. Freeman and Company, New York. Wachtmeister, H.F.E., Scott, L.J. and Perry, M.A. 1986. Encounters With Life: General Biology Laboratory Manual, 2nd Ed. Morton Publishing Company, Englewood, CO.

18

BACTERIOLOGY PART I: GENERAL LABORATORY PROCEDURES, BIOSAFETY and MORPHOLOGY Introduction: Up to this point, we have limited our examination of cell structure and function strictly to eukaryotic cells. In the next two labs we will focus our attention on prokaryotic cells. Prokaryotic cells differ from eukaryotic cells in several important features. Firstly, prokaryotic DNA is not packaged in a membrane bound nucleus but rather found in a region inside the cell called the nucleoid. The DNA in bacterial cells is primarily a single, circular chromosome whereas eukaryotic nuclear DNA is organized into discrete, linear chromosomes. The internal organization of prokaryotic cells lacks the elaborate membrane systems (e.g., endomembrane) and structures (e.g.: mitochondria and chloroplasts) that are typical of eukaryotic cells. Lastly, like plant cells, bacterial cells also have a cell wall; however, the composition of the bacterial cell wall is unique and contains the molecule peptidoglycan (a polymer of amino sugars). A primary feature of the microbiology laboratory is that living organisms are employed as part of the experiment. Most of the microorganisms are harmless; however, whether they are non-pathogenic or pathogenic (capable of causing disease), the microorganisms are treated with the same respect to assure that personal safety in the laboratory is maintained. Careful attention to technique is essential at all times. Care must always be taken to prevent the contamination of the environment from the cultures used in the exercises and to prevent the possibility of the people working in the laboratory from becoming contaminated. This care is referred to as aseptic technique. You will learn the appropriate procedures for manipulating bacterial cultures and will use these procedures to perform some basic microbiological techniques in the first part of this exercise. The second half of the exercise makes use of stains as well as well as immersion oil in order to view and make observations of bacterial morphology. The objectives of today’s exercise are thus to: • become familiar with using aseptic techniques to handle microorganisms. • gain experience in handling microorganisms by using fluorescein dye-labeled E. coli cultures to perform a series of exercises • illustrate the potential for contamination that is always present when working with microorganisms • learn basic microbiological techniques such as streaking for single colonies and inoculation of liquid cultures • provide an introduction to the Gram stain and allow you to practice using this differential stain • use light microscopy and oil immersion to assess bacterial characteristics such as size, shape, and association • encourage proper representation of bacteria using scientific drawings

19

As background for this lab, please read pages 534-535 in Campbell and Reece (2005). Please ensure that you have read over the guidelines on Safety (pages v-vi), and those on aseptic technique (Appendix D) prior to attending your lab. As well, you should become familiar with the contents of the University of Lethbridge Biosafety web site: http://www.uleth.ca/fas/bio/safety/biosafety.html

A. Introduction to Handling Microbes and Aseptic Techniques Procedure: Wear gloves and a lab coat for the entire exercise. 1. Tape bench coat onto the bench to cover your working surface. 2. Work individually over the bench coat and prepare a streak plate for single colonies using the following techniques: • Transfer (aseptically) a loop of the fluorescein dye-labeled E. coli culture provided to a sterile plate in the area shown by Figure 1a. • Once the first set of streaks has been made, flame the inoculating loop until red hot. Do not reintroduce the loop into the original culture. • Cool the loop by holding it in the region around the Bunsen burner flame for a few moments, and then make a second set of streaks as shown in Figure 1b, only crossing over the initial set of streaks once. • Flame the loop again, cool, and make a third set of streaks as shown in Figure 1c. Note, try not to gouge the agar while streaking the plate.

Figure 1: Procedure for inoculating a streak plate. Figure 1a

Figure 1b

Figure 1c

20

Label your plate and place it in the tray provided with the agar side up. Labeling plates of media: • Label on the agar side on the Petri dish itself (not on a piece of tape) • Label close to the edge of the dish following the edge • Do NOT write across the plate in big letters! 3. From the same fluorescein dye-labeled E. coli culture, inoculate one tube of nutrient broth using the following procedure. Again, the important thing to remember is that exposure of sterile liquids or bacterial cultures to air must be minimized. • Ensure that you have the tube of inoculum (microbe used for setting up a culture), inoculating loop and a sterile tube of medium available within easy reach. • Flame the inoculating loop until red-hot. Remove the cap from the tube of inoculum by grasping the cap between the last finger and the hand that is also holding the inoculating needle (Figure 2). Do not place the cap on the bench!!

Figure 2: Technique for manipulating test tubes aseptically.

• Flame the mouth of the tube by passing it rapidly through the Bunsen burner 2-3 • • • •

times. This sterilizes the air in and immediately around the mouth of the tube. Cool the loop on the inside of the tube, and then remove a loopful of the inoculum.. Reflame the mouth of the tube and replace the cap. Remove the cap for the tube of sterile medium in the same way, and gently place the inoculating loop into the medium. Flame the mouth of the tube, and replace the cap, and then flame the inoculating loop before replacing it on the bench.

21

• Note, when removing inoculum from a plate, cool the loop before picking up the bacteria. Work in pairs to complete steps 4 – 11. 4. Place a watch glass in the centre of the bench coat. 5. Obtain and label one Nutrient Agar (NA) plate with your names, date, organism tested and distance from the watch glass). 6. Place the agar plate on one side of the glass plate, either 5 cm or 10 cm from the watch glass (consult with the other pair at your bench to ensure that both distances are tested). Remove the lid from the plate and set aside (off the bench coat). 7. Using a pipette pump, and aseptic technique, draw up 1 mL of bacteria/fluorescein suspension. 8. Hold pipette tip 30 cm from glass plate and allow 10 drops to fall (one drop at a time) onto the glass plate. Put any remaining bacterial culture and the pipette into the disinfectant tray provided (be careful as this contains 10% bleach). 9. Put the glass plate into the disinfectant tray provided and place the cover back on the agar plate. Place on a tray on the side bench. 10. Use the hand-held UV lamps to inspect your bench coat, gloves, and lab coat. Caution: UV rays are harmful to skin and eyes. Ensure you wear proper eye protection. • What do you observe?

• What can you conclude about your techniques for handling microorganisms? How can you improve?

11. Your plates will be incubated for 16-20 hours at 37oC, and then refrigerated at 4oC. During the next laboratory period, count and record the number of colonies present (use Table 1). Table 1. Results from falling drop exercise Distance of plate from watch glass (cm) 5 10

Number of Colonies

22

• How far can droplets containing bacteria spray when a liquid culture is dropped?

23

B. Microscopic Characteristics of Individual Cells The small size of bacteria leads to a series of challenges involved with their study. Because living bacteria are almost colorless, they do not show enough contrast with the medium in which they live to be seen clearly under the microscope. Bacteria have a marked affinity for certain dyes, and when treated with these dyes they become readily visible. Their affinity for dyes is based on the fact that the cell wall has a net negative charge and therefore will attract positively-charged stain molecules. Observation of bacteria also requires a careful adjustment of the amount of light striking the specimen to increase the contrast between the cell and its background. Today, you will learn a special technique with the light microscope called oil immersion, which enables you to maximize both the magnifying power and the resolution of the light microscope in order to view very small objects. Work as a group of four students to complete the following exercise. You should review Appendix A (microscopy) prior to attending the lab. 1. Obtain a prepared slide of bacteria from one of the trays. Place the slide on the stage of the microscope, and focus with the 4X objective lens in place. 2. Once objects come into focus, switch to the 10X and then 40X objective. Remember that only the fine focus adjustment knob is required to bring the image into sharp focus when the higher power lenses are used. 3. Rotate the 40X objective away from the slide in preparation for using the 100X lens. Before clicking the 100X lens into position, add a single, small drop of immersion oil directly onto the slide surface at the spot where the light from the condenser is shining through. 4. Now rotate the 100X objective lens into place. Do not lower the stage; although it may appear that there is insufficient room for the 100X lens. Watch carefully from the side to ensure the lens slides into position. This lens will be sitting in the immersion oil. 5. Open the iris diaphragm as wide as possible to let more light into the field. Using only slight adjustments of the fine focus adjustment knob, bring the image into clear focus. Do not return the 40X lens back into position once you have immersion oil on your slide. 6. Note the shape of the bacteria. Typically bacteria come in three shapes, coccus (spherical), bacillus (rod-shaped), and spirillum (spiral-shaped) (Figure 3).

24

Figure 3. Examples of different bacterial shapes and associations commonly observed in culture. 7. Use the ocular micrometer (see Appendix C) to determine the size of the bacteria in your field of view. Typically it is the length of the bacteria that is used to indicate size. 8. Note the association of cells on your slide. The bacterial association refers to the relationship of different cells of the same species. Bacteria may occur singly or in pairs, chains or clusters (Figure 1). 9. Note whether your bacterial specimen has flagella. Some bacteria, but not all, have flagella that aid in locomotion. 10. Use the space below to make a proper scientific drawing of one of the four types of bacterial cells (coccus, bacillus-type, Spirillum volutans, and Proteus vulgaris) that are available for examination. Your drawing should depict the shape and association of the bacteria, while the drawing magnification will provide some insight on their actual size.

25

C. Gram Staining The Gram stain is a routine differential staining technique that aids in bacterial identification. A differential stain can be used to chemically distinguish between bacterial species that may be morphologically indistinguishable. Bacteria are generally divided into two groups, Gram positive and Gram negative, depending on their reaction to the staining procedure. The response of the cells to the stain is due to differences in the complexity and chemistry of the bacterial cell wall (Table 1). In the following lab exercise, you will prepare and stain two bacterial species to determine their size, shape, association, and Gram reaction. Table 1. Characteristics of Gram negative and Gram positive bacteria. Bacteria Type Characteristic Gram negative

Gram positive

Cell wall complexity

More complex

Relatively simple

Peptidoglycan content

Thin layer

Thick layer

Outer lipopolysaccharide wall layer Present

Absent

Stain displayed by cell

Crystal violet

Safranin

Cell color following Gram staining Red / pink

Dark purple

Work as a pair of students to complete the following exercise. 1. Wash your hands and disinfect the bench top using bleach. 2. Obtain broth cultures of Escherichia coli and Bacillus subtilis and label them using the tape provided. 3. Write the name of the bacterium to be stained at one end of the slide. 4. Flame an inoculating loop and keep it in your hand while it cools. 5. While the loop is cooling, open the tube of inoculum as you did in Part A. 6. Remove a loop full of culture; reflame the lip of the tube and replace the cap. 7. Spread the culture toward one side of your slide – the size of the smear should be at least the size of a dime.

26 8. Reflame the loop and set it aside. 9. Let the smear air-dry. You may use a warming plate or bench lamp if available. 10. The cells, although dry, will wash away when you apply liquid stain unless you first heat fix the cells. Heat fixation kills the cells and affixes them to the slide. Pass your slide five times through the flame to attach cells to the slide. 11. Place your slide on a paper towel, add one drop of crystal violet to the smear, and let the preparation stand for 30 - 60 seconds. 12. Hold your slide at the edges and gently rinse the slide with tap water for about 5 seconds. Be careful – crystal violet stains your fingers as well as the bacteria! (At this stage of the Gram stain, all bacteria will be colored purple regardless of their cell wall nature.) 13. Cover your smear with two drops of Gram’s iodine solution and let the preparations stand for 30 seconds. 14. Gently rinse the slide with water. 15. Holding the slide at an angle over the sink, gently trickle 95% ethanol (EtOH) onto the smear until very little dye washes off. Immediately gently rinse the preparations with water to prevent further destaining. 16. Apply one drop of safranin to your slide and let stand for 30-60 seconds. 17. Gently rinse the slide with water and remove any excess by gently tapping the side of the slide against a paper towel. Let both preparations air dry. 18. Observe your slide starting with the scanning objective (4X) and ending with the oil immersion lens (100X) of your light microscope. 19. Note the size (using ocular micrometry), shape, association, and Gram reaction of each type of bacteria in Table 2. 20. Tidy the bench and properly store the microscope. •

Are the species Gram positive or Gram negative? How do you know?

27 Table 2. Gram characteristic, size, shape and association of two bacterial species, B. subtilis, and E. coli, as determined following Gram staining and observation using a light microscope. Bacterium Bacillus subtilis Escherichia coli

Gram Reaction

Cell Size (µm)

Cell Shape

Association

28

Thought Questions: (Use the University of Lethbridge Biosafety web site: http://www.uleth.ca/fas/bio/safety/biosafety.html to help with answering questions 15) 1. What is an MSDS and where can you find one?

2. In Canada, the Laboratory Centre for Disease Control has classified infectious agents into 4 Risk Groups using pathogenicity, virulence and mode of transmission (among others) as criteria. What do these terms mean?

3. Lab facilities themselves are classified according to containment level. What does this mean? Provide 2 characteristics for containment levels 1, 2, 3, and 4. What containment level is your classroom?

4. What criteria would characterize an organism classified in Risk Group 1, 2 3 or 4? Provide an example of an organism found within each group.

29 5. There are many “Golden Rules” for Biosafety. Identify 4 common sense practices that will protect you in your microbiology labs.

6. a) What ultrastructural information about bacterial cells is revealed by the Gram stain?

b) Based on the Gram stain results, which bacteria were Gram positive?

c) Based on the Gram stain results, which bacteria were Gram negative?

7. List the four reagents used in the Gram stain and their respective purposes. Gram Reagent

Purpose

Literature Cited: Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San Francisco, CA.

30

BACTERIOLOGY PART II: ANTIBIOTIC-RESISTANT BACTERIA Introduction: In your lab last week you were introduced to general morphological characteristics of bacteria, and you also learned ways to manipulate them safely. In this exercise we are going to continue our studies of prokaryotes, but instead of concentrating on morphology, we are going to delve into aspects of their physiology and metabolism. Bacteria are adapted to survive in very diverse environments in which few other organisms could survive, including those with extremely high or low temperatures, or high amounts of acids or salts. Because of this tremendous adaptability, bacteria dominate the biosphere, making up at least 90% of the biomass on earth. They play a variety of roles in the natural world, but also have clear and direct impacts on human society as well. We have taken advantage of their genetic diversity (and hence, metabolic diversity) in a number of different ways, including bioremediation (the detoxification of polluted ecosystems), food production, mining, and more recently, have gained the ability to genetically modify them to overproduce compounds beneficial to human health. However, they also have negative impacts on our society. It is thought that bacteria are responsible for about 50% of all human diseases, including tuberculosis, cholera, anthrax, botulism and hamburger disease (caused by E. coli strain O157:H7). Currently, one of the most important issues society faces is the increase in the populations of microbes that have evolved resistance to antibiotics. This has led to our increasing inability to treat bacterial infections. Antibiotic resistance may arise naturally in bacteria, such that an antibiotic becomes ineffective in reducing numbers of bacteria of a particular species. Exposure of a population of bacteria to a given antibiotic will kill those that are sensitive to the antibiotic, but not those which have evolved resistance. Our society’s indiscriminate use of antibiotics further exacerbates this problem. Furthermore, genes encoding resistance to antibiotics are often located on plasmids; circular, double-stranded, replicable fragments of DNA that can be easily transferred to other bacterial cells, hastening the rate at which bacterial populations become resistant. Proliferation of antibiotic resistant pathogenic bacteria in our food supply over the past several years has also occurred. Antibiotic sprays are often applied to crop plants which encourages the proliferation of resistant microbes associated with crops and soil. The use of antibiotics in the livestock industry may lead to increased bacterial resistance in these animals, and these bacteria become integrated into soil via fecal deposition. The objectives of this exercise are thus to: • determine typical numbers of bacteria found on a common food source • determine what proportion of these bacteria are antibiotic resistant • apply the principles of the scientific method to analyze and interpret data collected

31 Please read pages 351 and 545-547 in Campbell and Reece (2005) in preparation for this exercise.

Week 1 Procedure: Please work in pairs to complete this exercise. Practice aseptic techniques when handling all lab materials. 1. One pair at the bench will test an unwashed vegetable sample; the other will wash their vegetable material first, and then follow the same procedures as for the unwashed material. Pairs responsible for washing vegetables should follow the directions provided by the container of vegetable wash at the side bench. Ensure that the pair working with unwashed material begins the exercise first! 2. Preparation of dilution series: • Collect four test tubes and four, 1mL pipettes from the side bench, and use the tape provided to label them with the dilution (10-1, 10-2, 10-3 or 10-4). • Weigh out 1 gram of vegetable tissue using the balance on the side bench. To obtain your material, use a razor blade to scrape off shavings from the outer surface of the vegetable material into a plastic weigh boat. • Use the spatula on your bench to transfer the shavings into the tube labeled 10-1. Add 10 mL of sterile water using the 10 mL pipette and pump provided (your instructor will show you how to use the pump). This represents a 1:10 dilution; in other words, 1 gram of tissue is now suspended in a total volume of 10 mL (or 0.1 grams of tissue per mL of water). • Vortex the contents of the tube vigorously for 30 seconds. • Use the 1 mL pipette labeled “10-1” to remove 1 mL of the solution from your 10-1 tube. Add it to your tube labeled 10-2. Add 9 mL of sterile water, and mix vigorously. This represents another 1:10 dilution. The 0.1 g of tissue transferred in the 1 mL from the 10-1 tube is now suspended in a total volume of 10 mL (or, 0.01 grams of tissue per mL of water) • Remove 1 mL of the solution from the second dilution tube (10-2) using the appropriately labeled pipette, and place it into the tube labeled 10-3. Add 9 mL of sterile water, and vortex vigorously. • Remove 1 mL of the solution from the third dilution tube (10-3) using the appropriately labeled pipette, and place it into the tube labeled 10-4. Add 9 mL of sterile water, and vortex vigorously. • What is the final dilution achieved? It may be easier to determine this by calculating how many mg of tissue are present per mL of water in the 10-4 dilution. 3. Plating: • Your instructor will demonstrate the plating procedure for you using sterile water to which vegetable material has not been added. This plate will act as a control.

32

• What is a control? Why was it necessary to incorporate a control into this • • • •

• • •

procedure? Collect three Nutrient Agar (NA) plates from the side bench and label them with your name, lab number, vegetable (indicate washed or unwashed), and the date. You will plate only your 10-2, 10-3 and 10-4 dilutions. Collect two Nutrient Agar + Kanamycin (NA + KAN) plates from the side bench, and label them as above. You will plate only your 10-2 and 10-3 dilutions. Use the 1 mL pipette labeled 10-2 to remove 0.1 mL of solution from your 10-2 tube. Remove the lid from the agar plate you labeled “10-2”, and add your 0.1 mL aliquot to the agar side. Remove a sterile glass spreader from the beaker at your bench, and use it to spread the drop of solution over the entire surface of your agar plate. Replace the lid on your Petri dish, and place the used spreader in the appropriately labeled beaker on the side bench. Repeat this procedure four more times, using a new sterile spreader each time. Once your five plates are prepared, cut a thin strip of Parafilm and wrap each of the plates. Place all five of your plates in an inverted position (agar side up) on the tray provided. Plates will be incubated at room temperature for 48 hours, and then will be stored at 4oC until your next lab period.

Week 2 Procedure: 4. Determination of total number of bacteria: • Collect your three NA plates from the tray at the side bench. Examine them for the presence of colonies. When examining plates, look for those that have between 30 and 300 colonies present. Any plates with fewer than 30 or more than 300 colonies are not useful from a statistical perspective, as there is more experimental error associated with these. • Count the number of colonies on the plate with between 30 and 300 colonies, and record the number of colonies and from which dilution they were obtained in Table 1 in your lab manual.

33 Table 1: Class data for the number of colonies growing on Nutrient Agar (NA) plates following the inoculation of 100 μL of a series of dilutions of unwashed and washed vegetable tissue and subsequent incubation at room temperature for 48 hours. Bench Number

Number of colonies on Amount of Dilution Total bacteria plate (between extract plated per gram of 30-300) tissue plated (μL)

1: unwashed 100 μL 2: unwashed 100 μL 3: unwashed 100 μL 4: unwashed 100 μL 5: unwashed 100 μL 1: washed 100 μL 2: washed 100 μL 3: washed 100 μL 4: washed 100 μL 5: washed 100 μL Control (water) 100 μL

•

Calculate the total number of bacteria per gram of original vegetable material. Remember that each colony is thought to represent progeny from one original bacterial cell. Record numbers in Table 1, and in the Table on the board. Use the equation below as a guide. Number of colonies Dilution *

x 10* =

total number of bacteria per gram of vegetable tissue

multiplying by 10 corrects for plating only 0.1 mL of your dilution onto your NA plate, and brings the number of bacteria up to the number of bacteria per mL of the dilution.

34 •

Prepare a bar graph showing the mean (+/- s.d.) total number of bacteria per gram of vegetable material versus the condition of the vegetable (washed and unwashed).

•

What conclusion(s) can you draw from these results with respect to numbers of bacteria found on unwashed and washed vegetable material?

5. Determination of the number of bacteria resistant to kanamycin: • Collect your two NA+KAN plates from the tray at the side bench. Examine them for the presence of colonies. Again, look for the plate that has between 30 and 300 colonies present, and count the number of colonies. Record the number of colonies and from which dilution they were obtained in the appropriate part of Table 2. • Calculate the total number of antibiotic resistant bacteria per gram of vegetable material. Record your numbers in Table 2, and in the Table on the board.

35

Table 2: Number of colonies growing on Nutrient Agar plus Kanamycin (NA+KAN) plates following the inoculation of 100 μL of a series of dilutions of unwashed and washed vegetable tissue and subsequent incubation at room temperature for 48 hours. Bench Number

Number of colonies on Amount of Dilution Total bacteria plate (between extract plated per gram of 30-300) tissue plated (μL)

1: unwashed 100 μL 2: unwashed 100 μL 3: unwashed 100 μL 4: unwashed 100 μL 5: unwashed 100 μL 1: washed 100 μL 2: washed 100 μL 3: washed 100 μL 4: washed 100 μL 5: washed 100 μL Control (water) 100 μL

36 •

Prepare a bar graph showing the mean (+/- s.d.) number of antibiotic resistant bacteria per gram of vegetable material versus the condition of the vegetable (washed and unwashed).

•

What conclusion(s) can you draw from these results with respect to numbers of antibiotic resistant bacteria found on unwashed and washed vegetable material?

•

Calculate means for total numbers of bacteria per gram of tissue from Tables 1 and 2. Record the mean values in Table 3, and use these values to calculate the percentage of antibiotic resistant bacteria found on your vegetable tissue.

37 Table 3: Percentage of kanamycin-resistant bacteria found on washed and unwashed vegetables. Vegetable tissue

Mean total number of Mean total number Percentage of kanamycin-resistant of bacteria/gram of kanamycinbacteria/gram of tissue tissue resistant bacteria

Unwashed: Washed: Control (water)

• What conclusions can you draw with respect to these data?

Literature Cited: Brock, D., Boeke, C., Josowitz, R. and Loya, K. 2004. Stalking Antibiotic Resistant Bacteria in Common Vegetables. The American Biology Teacher, 66 (8) pp. 554-559. Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San Francisco, CA.

38

CELL MEMBRANES: EFFECTS OF STRESS Introduction: Cellular membranes separate and organize chemicals and reactions within cells by allowing selective passage of materials across their boundaries. They are composed of a bilayer of phospholipid molecules interspersed with protein molecules. In addition, most membranes also contain very small amounts of carbohydrates that are usually associated with the phospholipids or proteins. Phospholipids are composed of a phosphate group, glycerol backbone, and 2 fatty acid chains (Figure 1). They are amphipathic; that is, each molecule has a hydrophilic (water-associating) region and a hydrophobic (water-avoiding) region. The charged (polar) phosphate group and glycerol group of each molecule are hydrophilic and the nonpolar lipid tails (fatty acids) are hydrophobic.

Figure 1. The structural formula (left) and symbolic representation (right) of a phospholipid molecule. When phospholipids spontaneously assemble in an aqueous solution, the most stable conformation is to have the molecules aligned so that the hydrophobic lipid regions turn inward and face each other, thereby avoiding contact with water (Figure 2). The polar phosphate head regions are arranged outwardly where they are in contact with water. Although the hydrophobic forces holding phospholipids in a membranous structure are individually weak and allow substantial movement of individual molecules, collectively they confer considerable stability to the overall structure.

39

Figure 2. Artificial membrane cross section depicting the orientation of phospholipids when exposed to an aqueous solution. Interspersed within the membrane are protein molecules. Each protein molecule is folded so that charged hydrophilic amino acid groups project into the aqueous phase inside or outside the cell and uncharged hydrophobic groups contact the inner lipid phase of the bilayer (Figure 3). As with any protein, relatively weak hydrogen bonds hold the membrane proteins in specific folded conformations. Within the membrane, proteins are not fixed in position but rather are free to move about. The proteins within the membrane perform a variety of functions including transport, enzymatic activity, signal transduction, and intercellular joining.

Figure 3. Lipid bilayer found in all biological membranes with embedded proteins. The physical and chemical integrity of a membrane is crucial for proper functioning of the cell or organelle of which it is a part. In lab two, we examined the selective permeability of artificial cell membranes and membranes from red onion cells. The permeability of a membrane is directly related to its phospholipids and transport proteins. In this exercise, we will continue our study of membranes by looking at the effects of various stresses (temperature and detergent concentration) on beet cell membrane integrity, and study how loss of membrane integrity leads to loss of membrane function. The roots of beets (Beta vulgaris) contain an abundant red pigment called betacyanin, which is localized almost entirely in the large central vacuole of beet cells. These vacuoles are surrounded by a vacuolar membrane and the entire beet cell is further surround by a plasma membrane. As long as the cells and their membranes are intact, the betacyanin will remain inside the vacuoles. However, if the membranes are stressed or damaged, betacyanin will leak

40 through the membranes and produce a red color in the water surrounding the beet tissue. The intensity of this red color will allow you to assess the damage produced by experimental treatments. The objectives of today’s exercise are thus to: • Continue to learn more about membrane structure and function by determining the effects of temperature and detergent treatments on membrane integrity and function • Become familiar with using a spectrophotometer to collect scientific data • Learn about scientific methodology, in particular, how to collect, present and analyze experimental results. Please read pages 99-101 and 124-130 in Campbell and Reece (2005) in preparation for this exercise. As well, review Appendix F and read chapter 9 in Pechenik (2007) prior to attending the next lab.

Procedure: Based on information provided to you in the lab manual and text readings, prepare hypotheses for the experiments investigating [a] the effect of SDS concentration and [b] the effect of temperature on beet membranes. [a] SDS:

[b] Temperature:

1. Working with your partner, use a cork borer with a 5-mm inside diameter to cut five uniform beet cylinders, and use a razor blade to trim each to 15 mm in length. Place all cylinders in a beaker and gently run cool tap water over them for two minutes to wash betacyanin from the injured cells on the surface. 2. One group at a bench will test the effect of various temperatures (see below), while the other group will test the effect of the detergent sodium dodecylsulfate (SDS). 3. For those testing SDS: • Add 6 mL of the appropriate concentration (see Table 1) to labeled tubes and place a cylinder of beet in each. • Let stand at room temperature for 20 minutes, shaking (gently) occasionally. • Use a dissecting needle to spear the cylinders and remove them quickly from the tubes, and then arrange the tubes in order from palest to darkest in colour. • Quantify the relative colour of the solutions, beginning with the palest and ending with the darkest (refer to Appendix D for information regarding use of a spectrophotometer). You will be reading the Absorbance at 460 nm. In order to set “100” on the percent transmittance scale, use the test tube containing the SDS solution (the reagent blank) which should be located next to the spectrophotometer. The light absorbance is a direct measure of the concentration of betacyanin and an indirect measure of membrane damage.

41 • Record absorbance readings in Table 1. • Rinse all test tubes and place in the baskets provided. 4. For those testing the effect of temperature: • Label five test tubes 1-5. • Gently, using forceps, remove two beet cylinders from the wash beaker, and touch each briefly to a paper towel to remove most of the adhering water. Place one in Tube 1 and one in Tube 2. Your lab instructor will collect Tubes 1 and 2 and place them in a freezer (-5oC) and refrigerator (5oC) respectively, for 15 minutes. • Obtain hot water from the bath at the side bench (caution: water is very hot) and mix in cold tap water (stirring with the stir stick, not the thermometer) until the temperature is 70oC. Select a third beet cylinder from the wash beaker and place it in the 70oC water for exactly 3 minutes. Then transfer it to Tube 5 and cover with 6 mL of distilled water at room temperature for 15 minutes. • Cool the beaker of water to 45oC by adding more tap water. Immerse a fourth beet cylinder for 3 minutes, place it in Tube 4 and add 6 mL of distilled water; let stand for 15 minutes. • Cool the water to 25oC and repeat the procedure for tube 3 with the final beet cylinder. • After 15 minutes of cold treatment, add 6 mL of distilled water to Tubes 1 and 2; let stand for 15 minutes. • After all beet cylinders have soaked for 15 minutes (no more – each cylinder will have to be timed on its own), remove them from the tubes. Use a dissecting needle to spear the cylinders and remove them quickly. • Arrange the tubes from palest to darkest in colour. Then quantify the relative colour of the solutions, beginning with the palest and ending with the darkest (refer to Appendix D for information regarding use of a spectrophotometer). You will be readingthe Absorbance at 460 nm. In order to set “100” on the percent transmittance scale, use the test tube containing the distilled water (the reagent blank) which should be located next to the spectrophotometer. The light absorbance is a direct measure of the concentration of betacyanin and an indirect measure of membrane damage. • Record absorbance readings in Table 1. • Rinse all test tubes and place in the baskets provided. A standard curve is used to determine the concentration of a substance. It is prepared by assaying various known concentrations of the substance you are trying to measure. In our case, we have measured the absorbance of known concentrations of betacyanin to make our standard curve (Figure 5). 5. Use the standard curve provided to determine the concentration of betacyanin that leaked from the beet cells for each SDS and temperature treatment (your instructor will demonstrate how to use the standard curve). Record these values in the appropriate column in Table 1 and on the black board. 6. Record your group’s betacyanin concentrations for the various SDS or temperature treatments in the table on the black board. Copy all of the class data into the appropriate spaces in Tables 2 and 3. Use this data to calculate the means and standard deviations of

42 betacyanin concentration for each SDS and temperature treatment (your instructor will provide you with formulae).

Betacyanin Absorbance (460 nm)

43

Betacyanin Concentration (μM) Figure 4: Standard Curve for known betacyanin concentrations.

Table 1. The absorbance (A460) and concentration of betacyanin leaked from beet (Beta vulgaris) cells following different SDS and temperature treatments. Tube #

%SDS

A460

Tube #

Temperature Treatment (°C)

Concentration (µM) 1

0

1

-5

2

0.025

2

5

3

0.05

3

25

4

0.25

4

45

5

0.50

5

70

A460

Concentration (µM)

44 Table 2. Class data for the concentrations of betacyanin leaked from beet (Beta vulgaris) cells following different SDS treatments as determined using spectrophotometry and a betacyanin standard curve.

Tube #

%SDS

1

0

2

0.025

3

0.05

4

0.25

5

0.50

Class Betacyanin Concentrations (μM)

Mean + s.d.

Table 3. Class data for the concentrations of betacyanin leaked from beet (Beta vulgaris) cells following different temperature treatments as determined using spectrophotometry and a betacyanin standard curve.

Tube #

Temp Trmt (°C)

1

-5

2

5

3

25

4

45

5

70

Class Betacyanin Concentrations (μM)

Mean + s.d.

45 Use the means and standard deviations from Table 2 to complete a scientific graph that demonstrates the relationship between the concentration of betacyanin released and the %SDS treatment.

•

Compare your results to your hypothesis for the experiment. Are your results as expected? Explain.

•

Based on the class results, what SDS concentration(s) were the most damaging to the beet cell membranes? Explain .

•

Based on the class results, what SDS concentration(s) had little or no effect on the beet cell membranes? Explain.

46 Use the means and standard deviations from Table 3 to complete a scientific graph that demonstrates the relationship between the concentration of betacyanin released and the temperature treatment.

•

Compare your results to your hypothesis for the experiment. Are your results as expected? Explain.

•

Based on the class results, what temperature(s) were the most damaging to the beet cell membranes? Explain.

•

Based on the class results, what temperature(s) had little or no effect on the beet cell membranes? Explain.

47

Thought Questions: 1. You probably noticed that there was some variability in the absorbance values that were obtained by different groups of students for the same experiment. What factors would affect the readings obtained from the spectrophotometers? (All machines are calibrated in the same manner and in good working order, so instrument error will not be an important factor.)

2. What is the advantage of using a spectrophotometer rather than your eyesight to measure color intensity?

3. In 1925, Gorter and Grendel obtained lipid from cell membranes by bursting and removing the contents of red blood cells. They spread the lipid in a layer one molecule thick on the surface of a tray of water. The area covered by this lipid layer was twice as large as the surface area they had calculated for the original cells. Can you explain the discrepancy?

4. You probably noted damage to membranes at both low and high temperatures. Is the mechanism of damage the same? If not, explain each.

48 5. After looking at Figure 5 below, explain in words how you think a detergent act on the molecules in the membrane.

6. In the experiment on the effect of temperature on beet membranes, name: a) the independent variable

b) the dependent variable

7. In the experiment on the effect of SDS concentration on beet membranes, name: a) the dependent variable

b) a controlled variable

Literature Cited Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San Francisco, CA.

49

ENZYMES Introduction: Without enzymes, most biochemical reactions would take place at a rate far too slow to keep pace with the metabolic needs and other life functions of organisms. Enzymes are catalysts that speed up chemical reactions but are not themselves consumed or changed by the reaction. The cell’s catalysts are proteins. As with all proteins, enzymes have a primary structure (a unique sequence of amino acids), a secondary structure (a folding or coiling of the chain), and a tertiary structure (a folding of the molecule into a complex three-dimensional structure). A combination of weak hydrogen bonds and stronger covalent bonds maintain protein structure. Enzymes speed up chemical reactions by lowering the activation energy required to complete the reaction; they are not consumed or changed by the reaction. An enzyme reacts with a substance known as a substrate to form a transient stage called the enzyme-substrate complex (Figure 1). The enzyme has a specific three-dimensional site, the active site, into which only one kind of substrate fits. Thus the enzyme and substrate relationship is very specific. The substrate remains in the active site for only a very short time before the product or products are released, whereupon another substrate molecule takes its place.

Figure 1. An enzyme and substrate interaction resulting in the formation of a product. Changes in temperature, alterations in pH, the addition of ions or molecules, and the presence of inhibitors all may affect the structure of an enzyme’s active site and thus the activity of the enzyme and the amount of product which results. Enzymatic activity can also be affected by the relative concentrations of enzyme and substrate in the reaction mixture. During the lab period today, you will investigate how changes in substrate concentration and temperature affect the enzymatic activity of catechol oxidase. You are familiar with the activity of this enzyme if you have ever seen brown spots or “bruises” on fruits and vegetables. The browning reaction is the result of the oxidation of phenolic compounds such as catechol to quinones, which subsequently polymerize into complex branched chains. Quinones are toxic to microorganisms and thus aid in the prevention of infection at a wound site. In normal undamaged cells, the catechol substrate molecules are maintained apart from the oxidizing enzyme molecules (catechol oxidase) by being sequestered in different parts or components of the cell. Following damage, the reaction proceeds as shown in Figure 2.

50

Figure 2. The reaction between the substrate (catechol) and enzyme (catechol oxidase), to form the product (benzoquinone). The objectives of today’s lab are to: • provide an introduction to enzyme structure and function • determine the effect of temperature and substrate concentration on enzyme activity • provide practice collecting, representing and interpreting data. As background for this lab, you will find it useful to read pages 77 - 85 and 150-157 in Campbell and Reece (2005).

A. Effect of Substrate Availability on Enzyme Activity In the presence of a large quantity of enzyme, the amount of product formed in a given period of time depends on the availability of substrate molecules. However, as more and more substrate is made available, at some point the initial amount of enzyme becomes limiting; that is, all the enzyme molecules are participating in reactions, and no matter how much more substrate is available, the amount of product formed in a given time period is the same. •

Based on the information provided to you in the lab manual and in text readings, prepare a hypothesis for the experiment investigating the effect of catechol availability on catechol oxidase.

Procedure: Work in pairs to complete this part of the exercise. 1. 2. 3. 4.

Label six test tubes in this sequence: 1, 2, 4, 8, 16 and 24. Using the pipet and pipet pump provided, add 5 ml of pH 7 buffer to each of the tubes. Add 1, 2, 4, 8, 16 and 24 drops of catechol to each of the appropriately labeled tubes. Add 23, 22, 20, 16, 8 and 0 drops of pH 7 buffer to the six tubes respectively to make all volumes of solution in the test tubes equal. 5. Add 30 drops of potato juice extract to each of the six tubes; shake to mix. The “juice” contains the enzyme catechol oxidase and is obtained by homogenizing potatoes in a highspeed blender. 6. Maintain the tubes at room temperature for 5 minutes, shaking each tube briefly at 1minute intervals. Don’t shake so hard that froth builds up at the top!

51 7. The yield of the enzyme-catalyzed reaction (i.e., the amount of product formed) will be proportional to the intensity of colour developed in each reaction mixture. After zeroing the spectrophotometer (Appendix D), record the intensity of colour of each solution after 5 minutes. Line up your six test tubes from palest to darkest in colour and pour each one in turn into the special spectrophotometer tube to read absorbance. Shake out residual liquid from the special test tube after each solution is measured; however it isn’t necessary to rinse the tube. In the instrument, light is absorbed by the benzoquinone molecules. Record your results and class results on Table 1. •

Why is it important to measure the absorbance of the palest liquid first, then the second palest, and so on?

Table 1. Class absorbance (A460) values for the substrate availability experiment. Drops of Substrate

Benzoquinone Absorbance (A460) Bench 1

Bench 2

Bench 3

Bench 4

Bench 5

Mean + s.d.

1 2 4 8 16 24

8. Work as a group to calculate the means and standard deviations for the different substrate treatments and place these in the appropriate spaces in Table 1. 9. Prepare a completely labeled graph (on the graph grid provided) that demonstrates the effect of substrate availability on the production of benzoquinone by the enzyme catechol oxidase.

52

•

Describe the trend (pattern) shown by the data.

•

Which treatment(s) displayed maximal product formation?

•

Do the results support your original hypothesis regarding substrate concentration and enzyme activity? Explain.

53

B. The Effect of Temperature on Catechol Oxidase As temperature increases, so does the movement of molecules. The rate of collisions between reactants (substrate and enzyme molecules) increases, and hence, the amount of product formed increases. Eventually however, the temperature reaches a point at which the weakest bonds maintaining the secondary and tertiary structure of the enzyme protein molecule are ruptured. The enzyme loses its catalytic function and is said to be denatured. Procedure: Work as a pair of students to complete the following exercise. 1. Label six test tubes with the temperature treatments to be used (3oC, 12oC, 20oC, 35oC, 50oC and 70oC) and add 3 mL of pH 7 buffer to each. 2. Place the test tubes in their respective water baths; set the 20oC tube on your bench. Allow 15 minutes for the buffer in each tube to temperature-equilibrate. 3. Add 10 drops of potato juice (containing the catechol oxidase enzyme) to each tube, without removing them from the baths, followed by 10 drops of catechol to each. Don’t forget to add potato juice and catechol to the tube at room temperature as well. Shake each tube, and allow them to incubate at their respective temperatures for 5 minutes, briefly shaking each at 1-minute intervals. 4. Move all of the test tubes (including the room temperature treatment) to the 3°C water bath and organize them from palest to darkest. Immediately remove the palest solution from the 3oC bath, pour it into the special spectrophotmeter test tube and read absorbance (see Appendix D for a review of spectrophotometer use). Do likewise with the other tubes, removing them one at a time from the cold bath, and ending with the darkest solution. The amount of product produced during a given period of time (the “yield) will be proportional to the intensity of colour developed in each reaction mixture. Record your results and class results on Table 2. •

Why did you move all tubes to the 3oC bath after their respective incubations?

5. Calculate the means and standard deviation for the different substrate treatments and place these in the appropriate spaces in Table 2. 6. Prepare a completely labeled graph on the graph grid provided to demonstrate the effect of temperature on the production of benzoquinone by the enzyme catechol oxidase.

54 Table 2. Class absorbance (A460) values for the temperature experiment. Temperature (oC) 3 12 20 35 50 70

Benzoquinone Absorbance (A460) Bench 1

Bench 2

Bench 3

Bench 4

Bench 5

Mean + s.d.

55

•

Describe the trend (pattern) shown by the data.

•

Do the temperature experiment results support your hypothesis? Explain.

56

Thought Questions: 1. At low levels of substrate availability, what limits the yield of an enzymatic reaction? At high levels of substrate availability, what limits the yield? At what point does the change take place?

2. Enzymes that lose their three-dimensional structure cannot interact with substrate molecules and thus lose their catalytic function; they are said to be denatured. The activity of catechol oxidase decreases with both high and low temperature. Does this mean that denaturation is occurring at both instances? Explain.

3. If you wish to preserve vegetables in the freezer, you must first boil them for a few minutes before packaging them. Why is this boiling step necessary? Wouldn’t the low temperature alone be sufficient to preserve the taste and appearance of the vegetables?

4. Is there an optimum temperature for catechol oxidase? Would all enzymes have the same optimal temperature? Explain why or why not.

57

5. How would your results differ if you had not shaken the tubes periodically during incubation?

Literature Cited: Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San Francisco, CA.

58

FERMENTATION AND THE SCIENTIFIC METHOD Introduction: Many metabolic reactions that occur in cells do not happen spontaneously, but instead require a source of chemical energy in the form of adenosine triphosphate (ATP). Typically cells manufacture ATP by oxidizing glucose using a series of enzymecatalyzed reactions. The oxidation of glucose takes place in two distinct stages: [i] glycolysis, where one molecule of glucose is converted to two molecules of pyruvic acid, a small amount of ATP and NADH. Glycolysis occurs in the cytoplasm of all living cells, and does not require oxygen to be present. [ii] aerobic cellular respiration, which consists of the Krebs Cycle and electron transport. These reactions occur in the mitochondria of cells, and oxygen is required as the final electron acceptor. Aerobic cellular respiration produces many more ATP’s per molecule of glucose than does glycolysis alone. If oxygen is not available or is not able to be utilized, then glycolysis becomes the major source of ATP production. In some organisms, like the yeast cells you will use in your experiment today, pyruvic acid is metabolized by another set of reactions termed fermentation. Fermentation results in the production of alcohol and carbon dioxide, but more importantly, oxidizes NADH back to NAD, enabling glycolysis, and hence, energy production, to continue. The fermentation reaction may be summarized as follows: C6H12O6 (sugar)

C2H5OH (ethanol) + 2 CO2 + energy

Yeasts are single-celled eukaryotic organisms related to molds and mildews. Under anaerobic conditions, yeasts break down sugars, releasing ethanol and carbon dioxide gas. Baker’s yeast (Saccharomyces bayanus) is a readily available organism that can be manipulated easily within a laboratory setting. We will use it in this exercise to explore what variables affect the process of fermentation. However, unlike other exercises you have completed thus far, it will be up to you to design and carry out an experiment illustrating some aspect of fermentation in yeast. You will use today’s period to think about what variable you want to test, to develop a hypothesis, and to design an experiment. Next week, you will carry out your experiment and collect and analyze your data. Thus, the objectives of this exercise are to: • Develop an understanding of fermentation and the generation of cellular energy • Allow students to gain experience in experimental design using a yeast system • Illustrate the Scientific Method through a hands-on approach Please read pages 160-162, 165-167 and 174-176 in Campbell and Reece (2005) and review Appendix F prior to attending your lab.

59

Week 1: Experimental Design Students will work in groups of four to design and perform an experiment. Your experimental design will be constrained somewhat by what materials we will be able to provide for you. The following reagents and equipment will be available for you to you carry out your experiment – please consult this list as you generate your hypothesis and design your experiment. • • • • • • • • •

Yeast suspension (7%) Sugar solutions: 0.5% glucose (all concentrations are in w/v), 0.5% sucrose, 0.5% lactose, 0.5%maltose, 0.1%, 0.2%, 0.3% and 0.4% glucose (all in water) 0.5% aspartame Waterbath at 40oC 15 mL Falcon tubes with holes punched in the lids (12 per bench) 500 mL beakers Tube templates showing level corresponding with volume Wax pencils Ethanol: 0%, 5%, 10%, 20% and 40% solutions containing 0.5% glucose

1. In order to assess fermentation, you will be measuring CO2 production by yeast over a 35 minute time period. To start, you should answer the following questions: a) What components are necessary in order for fermentation in yeast to occur?

b) What factors will affect the process of fermentation? (One place to start may be to think about the fact that as the reactions of fermentation are controlled by enzymes, the same factors that affect enzyme activity may also affect fermentation)

60 2. Choose a variable that you would like to test for its effect on fermentation in yeast (remember to consult the list of supplies available). Prior to going any further, you should confirm your choice with your Instructor.

3. Complete the worksheet found at the end of this exercise and hand it in by the end of the laboratory period. Note that you should be as detailed as possible as this will form the basis of your experiment next week. The following tips have been provided for you to help you design a workable experiment. Please consult these tips and incorporate the information into your experimental protocol (Page 2 of your worksheet).

Procedural Tips: • • • • •

• • • •

Add yeast (8 mL) to tubes first. Fill with sugar solution of choice (note, if testing a variable other than carbon source, use 0.5% glucose solution in water and a 40oC waterbath for incubation). Ensure that the volume of liquid in your tube is right up to the very top. Screw the cap on. Some liquid may come out of the holes in the cap – this is fine. Invert the tube gently to mix the contents. You will be placing your tubes in a 500 mL beaker filled with water that has been placed into the waterbath so that the beaker water is the appropriate temperature. You don’t want to place your tubes directly into any of the waterbaths in order to avoid contaminating the water with yeast cells. Place inverted tube into the beaker containing water in the waterbath. Start time = 0 immediately when you do this. At 5 minute intervals, lift the tube out of the waterbath, and mark the level of the solution in the end of the tube. Invert the tube to mix again and place back into the waterbath. Carry out readings for 35 minutes. At the end of 35 minutes, use the wax pencil markings to determine volume of CO2 produced at each time point. Use the tube template for small amounts.

Week 2: Experiment Collect your worksheet from your lab instructor and make note of any suggested changes to your experimental procedures. Carry out your experiment as designed, and collect your data. Use the data to prepare two figures, as described on the following pages.

61 1. Plot the change in CO2 production over your sampling times (this figure will give you information about the rate of the fermentation reactions).

•

Consider the rate of the fermentation reactions illustrated by your figure. You may notice that there is a lag initially (this is not an unexpected result). What may cause the lag in the fermentation rate?

•

Regardless of whether you saw a lag or not, how might you redesign your experiment to ensure that a lag does not occur?

62

2. Plot the independent variable you chose versus the total amount of CO2 produced.

•

Relate your results as illustrated by this figure to your experimental objectives (and to your null and alternative hypotheses). Do your results support your alternative hypothesis?

63

BIOLOGY 1010 FERMENTATION WORKSHEET Lab Number:

Date:

Group Members: ______________________________

______________________________

______________________________

______________________________

Hypothesis:

Independent Variable:

Dependent Variable:

Control(s)/Purpose of control(s):

Controlled Variables:

Supplies Required (only 12 tubes per bench are available)

64 Step by Step Experimental Summary (this will be the ONLY copy of the protocol so ensure that you can follow it!):

65

Thought Questions 1. Alcohol becomes toxic to yeast around a concentration of 12%. Why were you still seeing evidence of fermentation when you tested your cells with 20% ethanol?

2. How can the differential rates of fermentation using different sugar substrates be explained? Why did you not see fermentation when you used aspartame as a substrate?

3. Recognizing that fermentation reactions are catalyzed by enzymes, explain why we saw an increase in CO2 production as the concentration of glucose increased.

Literature Cited: Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San Francisco, CA. Black, S., Moore, R. and Haugen, H., eds. (2000). Biology Labs That Work: The Best of How-To-Do-Its, Volume II. National Association of Biology Teachers, Maryland.

66

DNA STRUCTURE AND FUNCTION Introduction: Up to this point in Biology 1010 we have studied, either directly or indirectly, the structure and function of three of the four major groups of macromolecules. In your exercise last week, we looked at how carbohydrates contribute to the generation of cellular energy. Earlier, we examined the structure of phospholipids and the role they play in maintaining membrane structure and function. We also studied enzymes, particular kinds of proteins whose function is to catalyze biological reactions. Our exercise this week will focus on the final group of macromolecules, known as the nucleic acids. There are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA and RNA are polymers consisting of many identical or similar monomers linked together in a chain. The monomeric units, termed nucleotides, in turn are composed of a grouping of [i] a pentose sugar, [ii] a phosphate molecule, and [iii] a nitrogenous base. A strand of DNA resulting from a chain of connected nucleotides is paired with another, complementary strand by way of bonds between the nitrogenous bases. As a result of the pairing, the double strand becomes helically twisted, and is referred to as a double helix. The genetic information in the cell is stored within the nucleotide sequence of DNA. This information is organized into discrete units called genes. DNA molecules do not exist in nuclei as linear double helices. In humans, each DNA molecule is made up of about 2 x 108 nucleotide pairs, which would be equivalent to a total length of about six cm (remember in humans that there are 46 such molecules in each nucleus, which represents about 2.8m of DNA!). A typical eukaryotic nucleus is only about 5μm in diameter, so the DNA must be packaged in order to fit. Proteins called histones have a net positive charge and thus bind to negatively charged DNA molecules to form a DNA-histone complex termed chromatin. When DNA is in this form it directs the synthesis of RNA (in a process called transcription) and in turn, RNA controls the synthesis of cellular proteins (translation). DNA is also the molecule responsible for the transmission of genetic information from one generation to the next. Although DNA is replicated while in the form of chromatin, the actual transfer of genetic information to subsequent generations occurs after chromatin becomes even more tightly condensed to form transient structures called chromosomes. Chromosomes are allocated in an organized and predictable manner to newly formed cells in a process called cell division. Although there are two different types of cell division that occur, mitosis and meiosis, we will focus only on mitosis in this exercise. Once mitosis is complete, chromosomes uncoil and remain as chromatin until the next round of mitosis occurs. The cell cycle is the term used to describe the series of events that occur in an actively dividing cell from the beginning of one cell division to the beginning of the next. In lab today, we are going to isolate chromatin from liver cells in order to get a sense of how much is present and what it looks like. In the second part of the exercise, you will learn about the events associated with the different stages of the cell cycle. You should be able to

67 distinguish between chromatin and chromosomes and when each is found within the cell cycle. The objectives of today’s lab are thus to: • Provide an introduction to nucleotides and nucleic acids, and to relate their structure to the functioning of these molecules • Provide students with the opportunity to extract and visualize DNA. • Learn about the cell cycle and where mitosis fits in • Learn how to prepare onion root tip squashes • Understand the stages of mitosis and what happens in each Please read pages 86-89, 293-298 and 359-361 in Campbell and Reece (2005) prior to attending your lab.

A. Extraction of Chromatin From Liver: In this portion of the lab exercise you will extract and view chromatin from non-dividing liver cells. First, cells will be ground and suspended in a saline (salt) solution. The next step is to release the chromatin from the cells – remember that membranes surround both the nucleus and the cell. Cell membranes are composed mainly of phospholipids and as such have an “oily” property. As dishwashers know, detergents “cut” grease. Likewise, sodium dodecylsulfate (SDS), a laboratory detergent, will cut or solubilize the oil-based cell membranes enabling extraction of chromatin. Once the membranes are solubilized (cells are said to be lysed), then chromatin can then be precipitated from the cell solution and visualized. Work in groups of four up to and including step six, then work in pairs. 1. Place a small amount of liver (about 1 cm by 2 cm) into a mortar and pestle. Chop the liver with scissors or a razor blade several times (for about 1 minute). 2.

Add 10 mL of 0.9% saline to the well-diced liver. Grind the tissue thoroughly (for about 5 minutes) with the pestle using a circular motion.

3.

To remove any unpulverized liver, strain the cell suspension through four layers of cheesecloth, into a small beaker. Rinse the mortar and pestle at this point to make clean-up easier later.

4.

Remove a drop of the cell suspension using a Pasteur pipet. Place the drop on a slide and next to it place a small drop of methylene blue. Use a toothpick to vigorously mix the two solutions on the slide. Add a cover slip and observe the cells with the 10X and then the 40X objectives. Identify the cells with their distinct, stained nuclei.

5.

Use a Pasteur pipet to add 10 drops of SDS to your cell suspension. Mix the suspension and remove a drop (with the pipet you used earlier for the cell suspension) to prepare another wet mount slide (clean and reuse the same slide each

68 time). Add methylene blue, mix together, add a coverslip and observe at 10X and 40X. 6.

Repeat Step 5 until membranes are no longer visible. This may require at least four to six additions of SDS. When you have determined that the majority of the cells have lysed, refocus using the 4X objective and remove the slide from the stage without altering the stage position.

7. Place a drop of cell suspension onto a clean slide. Do not add methylene blue or a coverslip but do spread out the drop so that it is a thin layer. Place the slide on the microscope stage and position it to view the drop. Do not alter the coarse focus; it should be in the correct focal plane from the last slide. 8.

As you view the preparation, your partner will add one drop of 95% ethanol (from the dropper bottle or with a clean Pasteur pipet) The ethanol will precipitate the chromatin and you should see masses of strand-like material ranging from thick to very thin.

9.

Because the preparation is uncovered, the alcohol-saline solution will evaporate quickly. Clean the slide and repeat Step 8 as you add the alcohol and your partner views the preparation.

10. Your preparation is not pure DNA. It contains protein and some RNA. If purification was your goal, you would have to remove these materials. As the first step, you would want to precipitate all chromatin from the cell suspension. To show how much is present use a Pasteur pipet to carefully add two or three pipets-full of alcohol, expelling it down the side of the beaker so that it forms a layer on top of the aqueous solution. You will see the chromatin appear as a white, mucus-looking substance at the interface between the solutions. Slow stirring will mix in the alcohol sufficiently to precipitate all of the chromatin. Centrifugation, re-suspension and washing, deproteination and chromatographic procedures would then have to be employed to purify the DNA. • In hindsight, what should you have done to prove that the nuclear and plasma membranes have to be ruptured before the chromatin can be extracted?

69

B. The Cell Cycle The orderly, complex series of events that occur in an actively dividing cell from the beginning of one cell division to the beginning of the next is termed the cell cycle. The cell cycle is divided into two major phases, interphase and the mitotic phase (M) (Figure 1). Interphase can be further divided into three stages: the first gap phase (G1), the S phase, and the second gap phase (G2). The G1 phase follows mitosis and cytokinesis and precedes the S phase. During this stage, a variety of growth processes such as gene expression and metabolism occur. The S phase is sandwiched between the gap phases and is when DNA synthesis and replication occur. Following replication, each chromosome is made up of a pair of sister chromatids held together at the centromere. The G2 phase follows the S phase and is a period of continued growth and initiation of activities in preparation for mitosis, the following step. Collectively, the three phases of interphase account for roughly 90% of the time spent in the cell cycle.

Figure 1. Stages of the cell cycle.

70 The other major phase of the cell cycle is the mitotic (M) phase, consisting of mitosis and cytokinesis. Mitotic cell division is necessary for the growth and repair of multicellular organisms. It is also a form of asexual reproduction for single-celled, eukaryotic organisms. Mitosis is nuclear division that results in two daughter nuclei, each with the same number and kind of chromosomes as in the nucleus of the parent cell. Mitosis is followed by cytokinesis, the cleavage of the cell cytoplasm into two halves each containing a nucleus. For convenience, mitosis is subdivided into five stages: prophase, prometaphase, metaphase, anaphase, and telophase (Figure 2). During prophase, the long thin strands of chromatin become condensed into discrete recognizable bodies called chromosomes. At this stage, the nucleoli temporarily disappear and the mitotic spindle apparatus begins to form in the cytoplasm. As the nuclear membrane fragments and disappears, the mitotic spindle becomes fully assembled during prometaphase. At the conclusion of metaphase, the chromosomes have aligned in an area that is equidistant from the spindle poles called the metaphase or equatorial plate. Anaphase is characterized by the splitting of the sister chromatids at the centromere followed by the migration of the recently separated chromosomes to opposite poles of the spindle apparatus. Telophase essentially reverses the early events of mitosis to form two, complete daughter nuclei: the mitotic spindle disassembles, two nuclear membranes reassemble, the chromosomes become decondensed and return to the long thin strands of chromatin, and the nucleoli reform. The completion of mitosis results in the formation of two separate daughter nuclei that have the same genetic composition as the parental cell. The first indication that cytokinesis is occurring can be viewed during telophase. In plant cells, a cell plate develops in the center of the cell. The cell plate enlarges until it joins with the plasma membrane at the perimeter of the cell. In animal cells, the initial indication of cytokinesis is the development of a shallow groove around the midline of the cell called a cleavage furrow. A contractile ring composed of microfilaments associated with the cleavage furrow is used to deepen the furrow and results in the cleavage of the original cell into two completely separate cells. Regardless of the nature of the cell, cytokinesis results in the formation of two daughter cells having identical nuclei and similar amounts of cytoplasm and associated organelles. Mitosis is easily studied using root tips from actively growing plants. In this lab exercise you will prepare onion root tip squashes and use prepared longitudinal sections of onion (Allium cepa) and broad bean (Vicia faba) to observe the various stages associated with mitosis. Although this part of the exercise emphasizes the events associated with mitosis, you should be able describe and identify all stages associated with the cell cycle.

71

Figure 2. Mitosis and cytokinesis in a typical plant cell. Preparation of Onion Root Tip Squashes: Work individually to complete this part of the exercise. 1. Label a microscope slide with your name. Using a razor blade, carefully cut off one onion root as close to the bulb as possible. In the plant root, cell division does not occur along the entire length, but only in one region near the tip. Place the root on your slide and trim the root so that you have a two mm root tip segment. Save the apical root tip and discard the remainder of the root. 2. Use an eye dropper to add two drops of 1N HCl to the root tip (be careful, acid will burn your skin). •

What is the purpose of adding HCl to your preparation?

72 3. Place your slide on the slide warmer (65°C) for five minutes. Do NOT let your preparation dry out during the incubation time. If required, add another drop of HCl to your preparation. 4. Use a paper towel to wipe away excess acid. Add two drops of toluidine blue stain to the root tip tissue. Continue to warm the slide at 65°C for three to five minutes, adding more stain if required. 5. Wipe away any excess stain and add one drop of fresh stain to your preparation. Cover with a cover slip and use a cork to press firmly, straight down (avoid lateral movement of the cover slip). This will separate and spread the cells. 6. Examine your preparation with the compound light microscope. With the 10X objective, you should be able to scan around and find cells in stages of mitosis, which can then be observed more closely with the 40X objective. Most of the cells will not be dividing, and their nuclei will be blue, often with one or more unstained nucleoli. Be patient and you will discover dividing cells. The chromosomes will again be coloured blue, and you ought to be able to pick up cells in all stages of division. If you are having difficulty identifying any mitotic stages, consult the Allium root tip or Vicia faba prepared slides available at the side bench. Share your good examples with your lab partner. •

Why did you observe so few cells displaying mitosis in your preparation?

•

Which mitotic phase did you see most frequently?

•

What features distinguish a cell in prometaphase from a cell in interphase?

7. Use the space below to sketch cells in each of the stages you observed.

73

Thought Questions: 1. Describe three structural differences between RNA and DNA molecules.

2. In the table below, name the five nitrogenous bases found in nucleic acids and indicate whether they are found in DNA, RNA, or both. Nitrogenous Base

Base Present in: DNA, RNA or Both

3. Compare (provide a similarity) and contrast (provide a difference) for the following pairs of terms: a) Chromosome and gene

b) Diploid and haploid

c) Centromere and chromosomal arm

4. What is a karyotype? How are chromosomes differentiated from each other?

74 5. Although interphase is not actually part of mitosis, recognition of its events is essential to understanding mitosis. Describe the events that occur during the G1, S and G2 phases of interphase.

6. Plot the amount of DNA in a cell as a function of time over the course of a complete cell cycle.

7. a) What aspects of cell division are common to both plants and animals?

b) What aspects of cell division are different between plants and animals?

8. Explain the advantages of having genetic material (genes) grouped into chromosome format, instead of all genes existing as separate entities.

Literature Cited: Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San Francisco, CA.

75

APPENDIX A: HOW TO USE THE COMPOUND MICROSCOPE

Your instructor will review the use of the compound light microscope with you. You may find it beneficial to label the parts discussed on the diagram provided (Figure 1).

Figure 1: The Compound Light Microscope Compound light microscopes have two sets of magnifying lenses, the ocular and the objective lenses. The ocular lens (eyepiece) is the lens that is closest to your eye. The ocular lenses on your microscope magnify objects to 10X their size. The set of lenses (typically four, the smallest being that with the least magnifying power, and the largest being that with the greatest magnifying power) closest to the specimen are the objective lenses; they are located on the revolving nosepiece. The body tube connects the two sets of lenses.

The arm and base of the microscope are the heavy cast metal parts of the microscope and are important support parts of the microscope that can be used to transport it. The arm supports a horizontal surface called the mechanical stage, upon which slides are placed for viewing. It is equipped with a spring-loaded clip that holds the slide in place, and two slide adjustment knobs used to move the slide on the stage. Place a slide on the stage and center it over the hole in the stage. Adjust the distance between the oculars to match your interpupillary distance (distance between your pupils). Revolve the nosepiece so that the lowest power objective lens (generally the 10x power lens) is in

76 position. To focus the microscope, locate the coarse and fine focus adjustment knobs at the base of the microscope, and use the coarse adjustment focus to move the slide close to, but not touching, the objective lens. Look at the stage from the side as you do this. On most microscopes this involves raising the stage, but on some the lenses are lowered. Also, on most microscopes an automatic stop will prevent you from moving the stage closer than about one centimeter from the lens. Now, look through the ocular lenses, and move the slide away from the objective lens until the specimen becomes clear (is in focus). Finish focusing with the fine adjustment knob. Once you have focused with the low objective power lens, you may switch over to the next higher power lens with only fine focus adjustments (the microscope is said to be parfocal). As you switch from one objective lens to another, you will notice that the working distance, the clearance between the lens and the stage, decreases with increasing lens power. This is illustrated in Figure 2 below.

Figure 2: The working distance (above) and the field of view (below) change with magnification of the objective lens.

It should be obvious to you why, when using the high power objective lenses (40X or 100X), you must use ONLY the fine focus adjustment knob; otherwise risk of contact and subsequent damage become high. Also shown in Figure 2 is the diminishing field of view (the illuminated area and contents that are seen when looking through the ocular lenses) as the objective lens power increases; this is due to a smaller and smaller aperture at the bottom of the lens through which light enters. This means that [a] things are harder to find on a slide when you are using high power since only a small fraction of the slide can be seen, and [b]

77 less light enters your eye and everything in the field appears darker. As a consequence, you will learn to [a] switch back to a lower power objective lens when you want to ‘scan’ around the slide, and [b] manipulate the amount of light coming into the lens using the iris diaphragm so that you can see the objects clearly. The amount and concentration of light coming through the specimen and hence to your eyes can be adjusted in several ways. First, of course, is the on/off light switch, generally located at the base of the microscope, and often associated with a rheostat to control light intensity. A condenser lens is mounted below the stage, and concentrates light on to the specimen; it generally needs no adjustment of position. An iris diaphragm is located below the condenser lens. Find the lever which controls the diaphragm; it can be very useful in adjusting illumination and contrast. Finally, some useful hints and cautions: • Never drag the microscope across the counter-top. Lift it with both hands by its arm, being careful not to tip it. • Use lens paper to clean glass slides and lens surfaces before using your microscope. • Water damages objective lenses. If water does contact a lens, wipe it off immediately. Also avoid getting water under the slide as it will stick to the stage. • If you have used immersion oil, use lens paper dipped in 60% ethanol to remove it from the 100x objective lens when you are finished. • Always start the focusing procedure with the low (10x) power lens. • When attempting to locate an object on a slide, remember that the image you see is reversed; that is, as you move the slide toward you on the stage, the slide is apparently moving away from you as you view it through the lens. • Some ocular lenses are equipped with pointers; they appear as a dark black line that will rotate if the lens is rotated in its tube. • When finished with the microscope, rotate the 4x or 10x objective into working position, lower the stage, turn off the light, wrap the cord so that it will not fall away when the microscope is lifted, and cover with the dust cover.

78 APPENDIX B: HOW TO MAKE A SCIENTIFIC DRAWING A scientific drawing is a graphical means of presenting results or observations. Guidelines for constructing a scientific drawing are given below. • • • • • • •

•

Although the cells are microscopic, your drawing should be larger in size. Use a sharpened, hard leaded (H or 2H) pencil, never ink or colored pencil crayons. Place the drawing slightly to the left side of the space, leaving room for labels to the right of the drawing. Always draw more than one cell in your drawing. This indicates the cellular association to an observer. (e.g.: found singly or as part of a tissue) The cellular detail of only one of the cells needs to be complete for labeling. Draw with one continuous line and do NOT retrace your lines. Do NOT shade in your drawing. Do NOT color your drawing. Place label lines horizontally (use a ruler to make them straight), with no crossed lines. Structure or organelle labels should be singular unless the label line branches to multiple structures (see examples below). • ‘mitochondrion’ is singular and ‘mitochondria’ is plural • ‘bacterium’ is singular and ‘bacteria’ is plural • ‘flagellum’ is singular and ‘flagella’ is plural Only draw and label structures that are visible in the field of view. Do NOT include structures that you know are present but are not visible or detectable with the light microscope.

A complete scientific drawing will always have a figure caption that is located below the drawing. The figure caption will contain three pieces of information: i) a figure number identifier (e.g.: Figure 1), ii) a brief description of what was observed and drawn, including the scientific name of the organism from which the specimen was taken and how the observation was made, and iii) the drawing magnification. The drawing magnification is a simple calculation that indicates the relationship between the size of your drawing and the actual size of the specimen. A diagram of a cell would be much larger than the actual cell, whereas a diagram of an elephant would be much smaller than the actual elephant. Magnification is defined as:

Size of drawing Actual specimen size

Where: - the size of the drawing is determined by measuring it with a ruler - the actual size of the specimen is determined by ocular micrometry - the number calculated has as many significant figures as the accuracy of your measurement (usually 2, if you measure in mm) An example of a proper scientific drawing is provided on the following page. Notice the positioning of the labels and figure caption and the type of information that is included in the figure caption.

79

Figure 1. A dividing Brachydanio rerio (zebra fish) cell in anaphase as observed using the light microscope. (1300X)

80 APPENDIX C: HOW TO DETERMINE THE SIZE OF A SPECIMEN METHOD 1: Use of an ocular micrometer. An ocular micrometer is simply an ocular lens that has a scale etched into the glass of the lens. A lens that has been so modified will have a red dot pointed on it. To measure the object you are viewing, simply replace your regular ocular lens with the micrometer lens. What you see is a scale overlying your object. By moving the stage, one end of the micrometer scale can be positioned at one end of your object. The scale is a line divided into tenths and each tenth is again divided into tenths. Each small division is called an ocular unit (ou). The scale and ocular unit graduations do not have any imperial or metric units assigned to them and so must be calibrated. The calibration of the ocular micrometer for the various objective lenses has been done for you (see Table 1).

Table 1. The calibrated values of an ocular unit for various Olympus light microscope objective lenses as determined by a stage micrometer. Objective Lens

Value of 1 Ocular Unit (mm)

4X (scanning)

0.026

10X (low power)

0.01

40X (high power)

0.0026

100X (oil immersion)

0.001

To calculate the length of the cell or structure you must always know the power of the objective lens used (so you know which conversion value to use) and the length of the object in ocular units. Therefore, if you were observing a bacillus bacterium with the 100x objective lens, and its length was 32 graduations of the ocular micrometer, the length of that bacterium would be 32 x 0.001 mm = 0.032 mm, or 32 μm.

81

APPENDIX D: ASEPTIC TECHNIQUE When working with bacteria, it is of utmost importance to practice certain aseptic techniques. This ensures that the culture being examined is not contaminated by organisms from the environment and that organisms being studied are not released into the environment. Although the bacteria used in these exercises are not pathogenic, it is still important to develop and practice good aseptic technique and work with care. •

At the beginning of the lab, wash your hands with soap and water, then wipe the lab bench with the disinfectant provided. Repeat these procedures at the end of the exercise.

•

Use a Bunsen burner to flame all nonflammable instruments used to transfer or manipulate bacteria. If you have long hair, tie it back. Long, floppy sleeved garments should be removed if possible or sleeves rolled away from your hands and wrists. When not in use, the Bunsen burner should be turned off.

•

Working with cultures requires efficient and careful work. Preread protocols so that you know what you need to do with the bacterial material. When culture tubes or plates are open, slant them away from you. Never lift a culture by the lid. Never lay culture caps or Petri plate tops on the bench and always recap cultures as soon as possible.

•

Always dispose of your prepared, used bacterial slides and plastic transfer pipettes in the disposal trays provided. Never place materials that have come into contact with bacteria in the broken glass box or common garbage.

•

If you spill a liquid culture on the bench top or floor, spray the area with disinfectant, place paper towels on the spill to contain it, and notify your instructor. The contaminated towels should be placed in the orange biohazard bag for autoclaving and disposal.

82

APPENDIX E: HOW TO USE A SPECTROPHOTOMETER Many procedures for the quantitative analysis of compounds in biological fluids are based on the fact that such compounds will selectively absorb specific wavelengths of light. For example, a solution that appears red to us (such as blood) absorbs the blue, yellow and green colors of light, while the red is reflected to our eyes. However, the eye is a poor quantitative instrument and what appears bright red-orange to one person may appear dull red-purple to another. A spectrophotometer is one instrument that will objectively quantify the amounts and wavelengths (kinds) of light that are absorbed by molecules in a solution. A source of white light is focused on a prism to separate it into its individual bands of radiant energy (Figure 1). One particular wavelength is selected to pass through a narrow slit and then through the sample being assessed. The sample, usually dissolved in a solvent, is contained in an optically selected tube or cuvette, that is standardized for wall thickness and has a light path exactly one centimeter across. These tubes are therefore expensive and must be handled carefully.

Figure 1. Representation of a photoelectric spectrophotometer. After passing through the sample, the selected wavelength of light strikes a photoelectric tube. If the substance in the cuvette has absorbed any of the light, the light transmitted out the far side will be reduced in total energy content. When it hits the photoelectric tube, it generates an electric current proportional to the intensity of the light energy striking it. By connecting the photoelectric tube to a device that measures electric current (a galvanometer), a means of directly measuring the intensity of light is achieved. The galvanometer has two scales: one indicates the percent transmittance and the other is a logarithmic scale with unequal division graduated from 0.0 to 2.0 indicating absorbance. Since most biological molecules are dissolved in a solvent prior to measurement, a source of error can be the absorption of light by the solvent. To ensure that the spectrophotometric measurement will reflect only the light absorption of the molecules being studied, a mechanism of ‘subtracting’ the absorbance of the solvent is required. To achieve this a reagent blank (the solvent) is first inserted into the instrument and the scale is set to read 0.0 absorbance (100% transmittance) for the solvent. The sample containing solute plus solvent

83 is then inserted. Any reading on the scale that is greater than 0.0 absorbance (less than 100% transmittance) is considered to be due to the absorbance by the solute only. The transmittance scale is a percent number; a ratio of the light exiting the sample tube to the light entering the tube. However, this number is not a linear reflection of the concentration of the solute molecules (Figure 2). The absorbance scale, on the other hand, does reflect a linear relationship. Although you do not necessarily know the exact concentration of the solute molecules in your sample, you do know that if the absorbance value doubles, the concentration of solute in your sample has doubled. Absorbance has no units, but the wavelength of the light is usually indicated using a subscript following the symbol A, e.g.: A580.

Figure 2. The relationship between percent transmittance and solution concentration (left) and absorbance and solute concentration (right). Instructions for Use of the Spectronic 20TM (see Figure 3): • • • •

The spectrometer should be turned on prior to the start of your lab period. If not, use the power switch (D, Fig. 3) to turn on the spectrophotometer, and then let it warm up for at least five minutes prior to reading your samples. Adjust the wavelength to the appropriate setting using the wavelength control knob (C, Fig. 3). Zero the spectrophotometer by adjusting the zero control knob (same knob as the power switch) so that the needle on the transmittance scale is set to 0%. There should be nothing in the sample compartment and the lid should be closed. Calibrate the spectrophotometer by inserting the reagent blank, a solution containing all of the components of the sample being measured except for the molecule of interest, into the sample compartment and closing the lid. Ensure that the cuvette is clean and dry before placing it in the sample compartment. Cuvettes must be oriented in the sample compartment such that the vertical line near the top of the cuvette is opposite the raised notch in the sample compartment. Rotate the absorbance control knob (E, Fig. 3) until the needle on the transmittance scale reads 100% (or 0 on the absorbance scale). Remove the reagent blank from the sample compartment.

84 •

Transfer your sample to a cuvette, insert it into the sample compartment, and read the absorbance value. Your absorbance reading should be between 0.0 and 2.0.

Figure 3. The Spectronic 20™ spectrophotometer. (A - Sample compartment, B Transmittance and absorbance scales, C - Wavelength control knob, D - Power switch / zero control knob, and E - Transmittance / absorbance control knob.)

85

APPENDIX F: SCIENTIFIC INQUIRY Background Reading As background for this material, you will find it useful to read pages 19 to 24 in Campbell and Reece (2005). You may also find the Science Toolkit (http://home.uleth.ca/bio/toolkit/) section regarding scientific method helpful.

The Scientific Process Today’s scientific methodology has its roots in the methodology of Aristotle. The scientific method consists of a progression of steps that include a preliminary information gathering phase (observations), the generation of a question based on initial observational data, the development of a hypothesis, the listing of a prediction of experimental outcome based on the hypothesis, the design of an experiment to test the hypothesis, the collection and analysis of experimental data, and the conclusions drawn from the experiment regarding the original hypothesis (Figure 1). During the experimental portions of the course, you will be asked to apply the scientific method, and know the terminology associated with it.

A. Hypothesis Observations of some natural phenomena lead you to formulate a question. When a question is asked, researchers attempt to answer it by proposing a hypothesis, a logical, testable, falsifiable explanation for the observed phenomenon. The hypothesis suggests an answer to the question. A hypothesis need not be extremely complex and should strive to be the simplest explanation. The data collected from an investigation cannot prove a hypothesis, but the data may support the hypothesis. Although a hypothesis can never be proven with 100% certainty (because we can never be certain that we have not overlooked an alternative explanation), repeated tests provide greater and greater support for a hypothesis, giving us more and more confidence that it is correct. Typically, scientific studies require two hypotheses that are mutually exclusive. For statistical reasons, we must contrast the hypothesis we wish to test, the alternative hypothesis (Ha), with a null hypothesis (Ho), a hypothesis in which we indicate that the treatment will have no effect on the variable that we are measuring. The null hypothesis permits us to make a statement that can be proven false by data.

B. Experimental Design The best way to test a hypothesis is with an experiment -- a replicated, controlled situation in which only one factor is manipulated. The factor being tested is varied in a known way, while all other factors are held as constant as possible. Critical to any experiment are aspects of experimental design. An experiment must be carefully designed so that appropriate materials are selected, all of the variables are identified,

86

Figure 1. Rigorous version of the scientific method. (In practice not all of these steps may be rigidly followed.)

external influences are controlled, and appropriate unbiased measurements are collected and analyzed. All of the variables with the experiment must be explicitly defined. In this course we will focus on experiments in which only a single factor is being examined at a time. The independent variable is the factor that is being manipulated or varied during the experiment; it is the cause. The dependent variable is the variable that shows some response or change to the manipulated independent variable; it is the effect. Controlled variables are factors that are held constant between treatments during the course of the experiment. All of the experimental treatments must be defined. A treatment is a test group of individuals subjected to the same levels or amounts of the independent variable. The experiment may also have a control treatment (please note this is NOT the same as a controlled variable!). The control treatment is a group in which the independent variable

87 is held at an established level or omitted. The control treatment serves as a reference for comparison of the experimental treatments and allows the researcher to determine whether the predicted effect is actually due to the independent variable. The experiment must be replicated (repeated) so that the conclusions are based on more than a single trial. This reduces the probability that the results we see are based just on chance and allows us to estimate this probability. The number of replicates used in each treatment is referred to as sample size, or n. The results from replicated experiments are usually averaged. Further analysis may be conducted using statistical tests. The type of data that you collect, how you will collect them, and how the data will be analyzed (the type of statistical tests you will use) must all be determined prior to conducting the experiment. Following a great deal of careful planning, the experiment can be carried out. The procedure is generally organized in a sequence of progressive steps. When conducting an experiment, precise notes should be taken and any exceptions or deviations from the original design must be noted.

C. Collecting and Analyzing Results The data recorded from an experiment may be collected as discrete data, in which each value represents a whole, separate, complete unit (e.g.: the number of bean seeds) or as continuous data, in which each value is taken from a continuous range using some type of measuring instrument (e.g.: bean seed mass). Raw data collected from scientific experiments are never presented in assignments, scientific papers, or published works. We must now include an assessment of these data by computing descriptive statistics, values such as the mean, which summarizes the central tendency and standard deviation or variance, which summarizes the spread of data (dispersion) around the mean. Descriptive statistics alone however will NOT permit us to directly compare the means and draw conclusions. The following formulae can be used to calculate the mean and standard deviation (however, most calculators will determine the mean and standard deviation for you):

Σ xi Mean (X) = n

where Σ(xi) is the sum of all individual, measured values (xi); n is the total number of measurements taken.

Standard deviation (s.d.) =

(Σ (xi2)) -

(Σ xi)2 n

n-1 where Σ(xi2) the sum of all individual squared measurements; Σ(xi)2 is the sum of all individual measurements squared; n is the total number of measurements taken. OR s.d. =

Σ(Xi - X)2 n-1

where Σ(xI - X)2 is the sum of the difference between each measurement and the mean squared; n is the total number of measurements taken.

88

D. Reporting Results The descriptive statistics from experiments are generally presented in tables or figures. Tables and figures serve two main functions. They are used to help you analyze and interpret your results and to enhance the clarity with which you present the work to a reader. Tables are columns and rows of numbers, whereas figures may be graphs, schematic diagrams, pencil drawings or even photographs. The format that you use to display your data depends on the data. If the data show a clear trend, a graph will accentuate and highlight this trend. If, however, the absolute data values are of importance then a table may be a better format choice for your data. Choose either a table or graph format to display your data but never show the same data in both formats. TABLES Tables are used to present results that range from a few to many data points. They are also useful for displaying the data from several dependent variables. For example, Table 1 shows the heart rate of frogs subjected to five temperature treatments. Notice that because each treatment had a different sample size (n) the sample sizes are also recorded in the table itself. The table is also accompanied by a table caption that appears above the table. The following guidelines will help you construct a table: • All values of the same kind should read down the column, not across a row. • The headings of each column should include the units of measurement • Each table presented is numbered consecutively (starting with Table 1) and is accompanied by a caption that is placed above the table. The caption briefly and clearly describes the content of the table. It makes reference to the organism (or cell, tissue type etc.) used in the study and its complete scientific name, the variables that were assessed, the treatments that were used, the types of values reported, and the sample sizes for the treatments. • Information and results not essential (e.g.: test-tube numbers, simple calculations) should NOT be included. • If the table is part of a report, the text must contain a least one reference to every table. Summarize the data and refer to the table; for example, “As the temperature increased from 5 to 25°C, there was a corresponding increase in frog heart rate from 10 to 80 beat per minute (Table 1)”. DO NOT write “See the results in Table 3.” It is not necessary to repeat each and every number in the table; refer to the trend only. GRAPHS Graphs permit trends and patterns that arise from a relationship between the independent and dependent variables to be emphasized. A graph is a visual summary of the results. Data may be represented in different formats depending on the type of data collected. You must determine whether your experimental variables (dependent and independent) are discrete or continuous. Discrete variables are those in which observations are placed into one of several mutually exclusive categories (e.g.: flower color, organelle type, inhibitor name, number of organelles with a cell). Continuous variables result from

89 quantitative observations in which the data may be any value in a continuous interval of measurement (e.g.: tissue sample weight, reaction time, amount of carbon dioxide exhaled, growing temperature, solution concentration). Table 1. The effect of temperature on the heart rate of Rana pipiens after a one-hour exposure to fresh water. The values represent the mean + the standard deviation; n is the sample size at each temperature. Water Temperature (°C)

Sample Size (n)

Hear Rate (beat min-1)

5 10 15 20 25

8 9 12 11 7

10 + 2 20 + 3 35 + 8 55 + 8 80 + 10

The following guidelines will help you construct a graph: • Use graph paper and a ruler to plot the values accurately. • Always print labels and use a sharp, hard-lead pencil. NEVER use a pen. • You must decide which variable is the dependent variable and which variable is the independent variable. The dependent variable is always placed on the y axis (vertical or ordinate axis), while the independent variable is graphed in the x axis (horizontal or abscissa axis) (Figure 3). • Label the axes with a few words describing the variable, and put the units of the variable in parentheses after the variable description (Figure 3). • The numerical range (scale) for each axis should be appropriate so that all data (including standard deviation or standard error values) can easily be plotted on the graph paper. Select your intervals and range to maximize the available graphing space. Choose intervals that are logically spaced and therefore permit easy interpretation of the graph (e.g.: intervals of 5s or 10s). Do not select intervals such as sixth, eighths, fourteenths, etc. If there are no data points at the low end of the scale, it is not necessary to begin the scale at zero. Otherwise you may have a large block of unused space on your graph. Similarly, it is not necessary to label every subunit of the scale; label every second, fifth, or tenth. It is important, however, that every subunit of the scale is of the same value. For example, you would not start with intervals of two at the lower end of the scale and then switch to intervals of ten at the upper end.

90

Y axis Dependent variable label here (variable units)

25

Legend: = high dose = low dose

20 15 10 5 0 0

15 20 25 30 X axis Independent variable label here (variable units) Figure 3. This is the figure caption. It always appears below the figure and describes the graph. You must include all variables, treatments, types of values reported, symbols, sample sizes amd organism, tissues or cell studied in the figure caption.

•

•

•

•

•

5

10

35

Several data sets can be placed on a single graph, however they must be clearly labeled, usually by a legend, and the points of each data set should be given different symbols. Colored graphs are rarely used in scientific publications. Select a graph type that best describes your data. Line graphs, bar graphs and scatter graphs are most frequently used in scientific presentations. The choice of graph type depends on the nature of the variables being graphed. (Please see below.) All graphs are given a caption which is located below (not above) the graph. Each graph is labeled as a Figure (NOT graph) and given a number (e.g.: Figure 1). The first figure referred to in the text is Figure 1, the second, Figure 2, and so on. A brief description of the information shown in the graph (all variables, treatments, types of values reported, symbols, sample sizes, and organisms, tissues or cells studied) follows the figure number. Capitalize the first word in a figure title and place a period at the end (Figure 3). If the figure is part of a report, the text must contain at least one reference to every figure. Summarize the data and refer to the figure. For example, “The 0.01 µM solution of the plant growth substance promoted plant height in canola (Figure 4)”. DO NOT write “The results are shown in Figure 4”. It is not necessary to repeat every data point shown in the figure, but do refer to the trend. Computer-generated graphs may be used instead of hand-drawn graphs provided that they conform to the proper format. If you do not know how to manipulate the

91 various attributes of the graph to generate a properly labeled graph, it may be advantageous to hand-draw your graph. Line graphs are generally used when the independent variable is continuous, one or two dependent variables are reported, or several data points are reported. Line graphs show changes in the quantity of the selected variable and emphasize changes of the values over their range. Figure 4 shows the relationship between the application of two concentrations of a substance to canola plants during a 30 day incubation period. The following guidelines will help you construct a line graph: • Follow the general guidelines for making a graph. Be sure to decide on uniform scales for each of the axes. • Plot the data as separate points. Use separate symbols to differentiate between different data sets. Be sure to define these symbols in your legend. • Deciding whether to connect the data points on a line graph depends on the type of data and how they were collected. To illustrate trends, draw smooth curves or straight lines to fit the values for any one data set. To emphasize meaningful changes in value on the x-axis, connect the points dot to dot. Do not extrapolate beyond a data set unless you are using it as a prediction technique because you do not know from your experiment whether the relationship holds beyond the range tested. Do NOT force the lower end of the line through the origin (0, 0).

80

Plant height (cm)

60 40 20

0 µM (control) 0.01 µM

0 0

5

10 15 20 25 30 Days post-application Figure 4. The influence of gibberellin, a plant growth substance, on elongation (plant height) of Brassica rapa plants as measured over a 30 day period following application. The values represent the means + s.d., n = 25.

35

92 Bar graphs are usually used to represent data that represent discrete or discontinuous groups or non-numerical categories, thereby emphasizing differences between groups. Bar graphs may represent frequency data, that is, data in which measurements are repeated and the counts are reported (Figure 5) or data from discrete groups that do not lend themselves to a linear scale (Figure 6).

8

Frequency

6 4 2 0

1

2

3

4

5

6

7

8

9

Number of Golgi bodies observed Figure 5. The frequency of Golgi body occurence with Chinese hamster kidney cells as determine using transmission electron micrographs, n = 21 cells.

Cell Size µm)

2000

1500

1000

500

0 coli E.E.coli

Erythrocyte

Lymphocyte

Sperm cell

Human egg

Amoeba

Cell type Figure 6. Comparison of the sizes of various cell types as determined from literature values. The values shown represent the means + s.d., n = 20 cells for each type.

Frog egg

93

E. Communicating Results Effectively communicating scientific results is just as important as designing and carrying out the experiments. Scientific findings must be shared and may be communicated either in oral (spoken presentations) or written (published papers) fashion. A scientific paper will consist of the following components: TITLE (a statement of the question posed or nature of the investigation) ABSTRACT (a short summary of all components of the paper) INTRODUCTION (a brief literature review of the question being addressed and statement of the hypothesis being tested and resulting predictions) MATERIAL AND METHODS (a brief summary of what was done and how) RESULTS (presentation of data in tabular or graphical format and accompanying notation of trends and patterns observed) DISCUSSION (interpretation and discussion of results) LITERATURE CITED (alphabetical listing of all references used)

Literature Cited Campbell, N.A. and J.B. Reece. 2005. Biology, Seventh Edition. Benjamin Cummings, San Francisco, CA.

94

APPENDIX G: CONVERSIONS AND TAXONOMY Metric Conversion Values Below is a list of metric conversion values that you must be familiar with. If these are new to you, please memorize them. Metric conversion values will NOT be provided for quizzes and exams. Volume 1 L (litre) = 1000 mL (milliliter) 1 mL = 1000 µL (microliter) Length 1 m (metre) = 100 cm (centimeter) 1 cm = 10 mm (millmeter) 1 mm = 1000 µm (micrometer) 1 µm = 1000 nm (nanometer) 1 nm = 1000 pm (picometer) Mass 1 kg = 1000 g (gram) 1 g = 1000 mg (milligram) 1 mg = 1000 µg (microgram) Basic Taxonomy Typically in biology the binomial name (a two part Latin name) and not the common name is used to refer to an organism. The first word in the binomial designates the genus to which the organism belongs. Both words together refer to the species name. For example, you examine a micrograph showing rat liver cells. The rat belongs to the genus Rattus. The first letter of the genus name is always capitalized and the entire word is italicized. There are several species of rat that belong in this genus, but the specific organism from which the cells came from was Rattus norvegicus. You should take note of how the species name is represented. Scientific convention requires that if a species name is typed, it should be placed in italics. Since it is difficult to discern italic hand writing from normal hand writing, hand written species names are always underlined (e.g.: Rattus norvegicus). As above, the first letter of the genus name is always capitalized. However, the second part of the species name is an adjective that describes the genus and it is never capitalized and always appears in lowercase letters. You should get in the habit of properly using the binomial name whenever possible.