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Biology 05LA Fall Quarter 2013

Introduction

Prologue
This is the first installment of the Biology 05LA LAB MANUAL. While this document does not contain specific descriptions of lab activities, it does provide a great deal of information that is essential to the student wishing to do well in the course. Your first assignment for the quarter is to print a copy of this information and to read it carefully. Keep in mind that you will be held responsible for any information presented in this document! Therefore, you should keep a copy available for later reference. The remaining portions of the lab manual, which comprise the individual lab exercises, will be published on the course websites (either 05A or 05LA). You will be expected to have printed a copy of each lab exercise before your lab meeting and to have done the required preparation for that lab as described later in this document. Dont forget to bring a copy of current the lab exercise to your scheduled lab session. Do not use copies of the Biology 05LA lab manual from previous quarters serious consequences may result.

Table of Contents
Item 1. Laboratory Schedule Page # ii Content This lists the dates and times for the different lab activities and also gives the point breakdown for each graded item in the course This provides information about lab preparation, something about the different kinds of labs that will be presented, our expectations about performing and completing the lab exercises, and some commentary about the content and preparation for the lab comprehensive exam. This describes our view of what constitutes academic dishonesty and our policies for dealing with these issues. This describes what you need to do if you miss a lab. This form is to be used if you missed a lab and expect to be excused for this absence. This describes the graded library exercise that is part of the course curriculum. This describes several important lab safety issues.

2. Introductory Comments

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3. Academic Integrity Statement 4. Lab Absence Protocol 5. Excused Absence Request Form 6. The Library Exercise 7. Laboratory Safety

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Biology 05LA Fall Quarter 2013

Introduction

Laboratory Schedule Biology 05LA - Fall 2013


Week 1 2 Dates
9/30 10/7 Graded Item or Item Due 1. Introduction to Microscopy and Attendance is required or you Practical Microscope Use. will be dropped from the class

Lab Topic

Possible Points

2. Spectrophotometry & Quantitative Data Analysis. 3. Diffusion, Osmosis and the Permeability of Cell membranes. 4. Lab 4A Genetic Analysis (Part I) and Lab 4B Hypothesis-Based Inquiries & Intro. Writing Clinic

Lab Quiz lab 1 + prep. for lab 2.


Start Library Exercises

10/14

Complete in-class worksheet Lab Quiz labs 2&3 and prep. for lab 4 and 4A.
Note: Please bring your texts to lab for reference.

10

10/21

Complete in-class worksheet

5 20 30 10

10/28

5. Enzymes.

Enzyme Introduction due @ lab start.* F. & R. Intro. Due @ lab start.*

11/4

6. Fermentation and Respiration.

Hand in a Lab 5 worksheet

Veterans Day Holiday


7 8 9 10 10
11/11 (no labs scheduled)
End Library Exercises.

11/18

7. Genetic Transformation (Part I)

Hand in a Lab 6 worksheet

10

11/25 12/2 12/6

Thanksgiving Holiday
(no labs scheduled) 8. Genetic Transformation and Genetic Analysis (Part II) Lab Final Exam *** 7:10-8:00pm Complete in-class worksheet Rooms TBA Library Exercises Total Lab Points** 10 80 15 200

* Introductions to lab write-ups are due at the beginning of the lab period. Late papers will not be graded. ** Two extra credit assignments will be offered this quarter. These points will not affect the points total and no student can have more than 200 points. ***Lab Exam: If you have a conflict of a non-academic nature (an evening job, for example), then you must negotiate an accommodation with your employer or the instructors by 11/8, or withdraw from the course.

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Biology 05LA Fall Quarter 2013

Introduction

INTRODUCTORY COMMENTS
"Much of our educational system seems designed to discourage any attempt at finding things out for oneself, but makes learning things others have found out, or think they have, the major goal. It is certainly true that it is easier to teach a set of facts than it is to encourage an inquiring mind to make its own discoveries. Once a student has learned that he can find things out for himself, bad pedagogy is probably only an irritant." Anne Rowe; The Making of a Scientist All too often laboratory exercises are dull and lifeless because of the constraints imposed by funds for supplies and equipment, by student numbers, and by space. When choosing and designing the experiments for Biology 05LA students, we have picked exercises that stimulate their appreciation for cell and molecular biology and engage them in the processes of investigation and critical thinking. Also relevant is the revolution in cell and molecular biological approaches that has developed in the past 2 decades. Not only has the way of thinking about Cell and Molecular Biology issues changed, but the rate at which our knowledge is being accumulated has increased dramatically. We feel that these changes make it imperative that we emphasize the processes by which knowledge is generated and changed. In addition, it is our hope that we encourage the students to actively participate in these exercises rather than passively absorb facts and figures. A concluding thought. This manual is presented as a guide for your efforts in lab. In order for it to have its intended outcome, you must read it carefully and think about what you read. This thought is summarized in the sage words of an emeritus faculty member of this department: You cant read this stuff like youre reading a comic book!

The Laboratory Examination:


A comprehensive laboratory examination is part of the course curriculum. It will be given week 10 (12/6/13) - further details regarding the exam are given in lab. A large proportion of your BIOL 5LA course grade will be based upon your performance on this exam. Therefore, you must do well on this examination if you expect a B or an A in the course. The coverage of the exam is outlined, in part, in the Learning Goals/Desired Outcomes sections that appear at the end of each laboratory exercise. These clearly and explicitly state what is expected of you for each of the labs. You should refer to these listings both before and after each of the laboratory exercises. You should be able to prepare written responses (in your lab notebook) for most of the listed items. Do this as soon as possible after you have completed each lab. These responses should be written at the highest level possible with the available resources; your lecture notes, the Campbell text, and the lab manual. Further, you should review these responses periodically in the time between when they were written and the exam. Your TA has been instructed to monitor your progress in this activity and to help you in this effort. The exam will also cover topics that should be a part of the write-ups that you will prepare for the two experimental labs.

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Introduction

Record Keeping and Some Suggestions about Lab Preparation


The laboratory notebook is your record of your work. As mentioned above, you will be examined on all laboratory exercises performed this quarter. The laboratory notebook will become a valuable resource for preparation for these evaluations. You are encouraged to keep a record of your laboratory activities in a manner that will make your notebook entries useful for later study. Some suggested guidelines follow. The labs for Biology 5LA may be classified into three basic types: tool box laboratories, investigative laboratories, and the tutorial laboratories. Each type has somewhat different requirements for adequate preparation and record keeping. 1) The toolbox laboratories. Labs 1 & 2 fall into this category. These exercises are so named because they are intended to provide some basic laboratory skills and/or conceptual tools that are required for Biology 5LA and many other science courses that you will be taking here at UCR. a) Before your lab period, you should carefully read the entire lab exercise in this manual including the Learning Goals/Desired Outcomes sections that are posted at the end of each lab exercise. Once this is done you should write down (in a sentence or two) the purpose of each part of the days lab exercise. b) During the lab period, you should describe all of the observations you have made or the techniques you have learned in your lab notebook. For the former, accurately labeled and scaled drawings or sketches will often serve this purpose. It might be necessary to accompany these drawings with some verbal commentary when there are features of your subject that do not lend themselves to being recorded in the form of a diagram. For the latter you should focus on the aspects of the technique that you found particularly difficult or different than what you had expected. c) After the lab period, you should make sure that you can adequately respond to every item on the Learning Goals/Desired Outcomes listings found at the end of each lab. Further, you should write down any problems you had with the techniques, the observations, and etc. If you had such problems, you should attend your TAs office hours so that you can discuss them with your TA. 2) The investigative laboratories. Labs 5 & 6 fall into this category. These are open-ended experimental labs that are truly investigative in nature. In addition to being instructional about particular subject areas of the course, these laboratories are also intended to familiarize you with the process of Hypothesis-Based Science (a thorough discussion will be presented in Lab 4). a) Before your lab period, you should prepare the Introduction for the days lab exercise following the instructions presented in Lab 4. A copy of this introduction will be handed for grading in on the day that you are to perform the laboratory exercise*. You should retain a second copy of your introduction for later use. You should also prepare, in advance, all the necessary data tables you will need for the current weeks exercise. This should be done in the lab notebook.
*Note: introductions received after 20 minutes past the starting time of your lab session will receive no credit. Also, you must perform the lab exercise to receive credit for its introduction.

b) During the lab period, your focus should be on producing the best possible data. This is accomplished by executing the lab protocols exactly as described in this manual and by your TA. If you have any doubt about what is expected of you, please get these doubts clarified before you produce useless and unintelligible data. You should also be very careful to record your results in a retrievable manner so that they can be used to complete your reports. iv

Biology 05LA Fall Quarter 2013

Introduction

c) After the lab period, you should prepare the results and discussion sections of your reports following the instructions presented in Lab 4 in this manual. The results and discussion sections of your reports will be completed in your lab notebooks. For the Enzyme and Fermentation-Respiration labs, you will hand in a copy of the processed data for these labs at the times specified in the lab schedule. You should also make sure that you can adequately respond to every item on the Learning Goals/Desired Outcomes lists found at the end of these labs. 3) The tutorial labs. Labs 4 and 7 fall into this category. These labs are intended to complement and underscore the lecture coverage as well as provide some additional experience in this area that should broaden your understanding of the topic. a) Before your lab period, you should review the lecture notes and the text coverage of the subject areas for that days lab. You should also carefully read the exercise in the lab manual. For lab 7, you should complete the flow chart for the transformation experiment you will be performing. b) During the lab period, you should participate in a manner comparable to that described for the preceding lab types. c) After the lab period, you should make sure that you can adequately respond to every item on the Learning Goals/Desired Outcomes lists found at the end of each lab. For the transformation lab you will need to return to the lab at a time specified by your TA to count your plates and then use these counts to calculate the transformation efficiency for the experiment you performed. We hope that this introduction to your laboratory makes you aware of what is expected of you. Hopefully, we will have convinced you that you need to be an active participant in order to take advantage of the wealth of theoretical and practical information to which you will be exposed over the course of the quarter.

Biology 05LA Fall Quarter 2013

Introduction

Academic Integrity Statement


Cheating Of Any Kind Will Not Be Tolerated. Each student is expected to demonstrate honest individual scholarly achievement in every examination, in every written assignment, and in conducting all laboratory exercises. If you are repeating the course, you must write entirely new lab assignments this quarter. It is not acceptable, and regarded as cheating in this class if you reuse all or parts of lab assignments from previous offerings of this course. It is also considered cheating for first-time students to

plagiarize lab reports from the present or earlier course offerings. During the exam and the lab quiz, all notes and books must be closed and placed out of view. Every student located near open notes or books during any part of the exam or quiz will receive zero credit. For the comprehensive exam, students must sit in assigned locations in the exam room, and may be required to show their student ID cards to their TA. Any student involved in any form of cheating could be dismissed from the class, assigned a failing grade, and recommended for disciplinary action. Also, a note concerning the dishonest activity will be added to the individuals student record.

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Biology 05LA Fall Quarter 2013

Introduction

Lab absence protocol:


Laboratory attendance is mandatory. You must go to your assigned lab each week and arrive on time. All assignments are due at the beginning of the lab period, but we will accept assignments that are handed in within the first 20 minutes of your scheduled lab period. Assignments handed in after the first 20 minutes will not be graded. You should always be on time for lab because important information is always presented at the start of each lab. If you are unable to attend your regularly scheduled lab because of illness or some other dire circumstance, you will need to file an excused absence request form (found on the next page) and associated materials well before the start of the lab you will miss OR within 24 hours of the end of the period of any medical or other excuse that you may have for your absence. Along with this form, you must submit the following: The original copy of your excuse. No photocopies will be accepted! Any assignments that may have been due for the lab period that you missed.

The place for submitting these documents will be the drop box located on the office door of the academic coordinator for the course. For this course the lab coordinator is Dr. Laura Abbott who is located in 1218 SPTH. Any conflicts of a religious nature should be brought to Dr. Abbott by the second week of class. Upon receipt of the above, the academic coordinator will make a decision as to the status of your request and will notify you, via email, of her decision. Possible responses include: 1. Your request is accepted. In this case the academic coordinator will try to schedule you into another lab session occurring later in the week (your class schedule will be checked to make sure that you can attend). If rescheduling is impossible, your scores for any quizzes or other in-class, graded items will pro-rated. You will need to see coordinator to arrange a method whereby you can complete any out-of-class assignments associated with work related to the missed lab this arrangement must be made within one week of the missed lab. If an assignment was submitted with your request, it will be accepted. 2. Your request is denied because we are not able to corroborate your excuse OR your excuse is considered inadequate. In this case, we will not accept any assignments that were due on the day of the missed lab, you will not be reassigned to a lab occurring later in the week, and you will not be allowed to complete any assignments associated with work related to that lab. 3. Your request is denied because it was not submitted in the correct manner or within the time period described above. Please note: Filing a request for an excused absence does not guarantee that it will be accepted. Excuses for medical appointments, funerals, accidents, and educational conferences are usually accepted - however it is unlikely that more than one excused absence would be granted in a quarter. In other words, if we accept one excuse for a missed lab, it is unlikely that we will accept a second. An absence from your lab means that you miss information we deem critical for earning credit in this course. In cases where a student misses more than two labs, the student will need to withdraw from the course or will receive an incomplete should that students completed work be of passing quality. vii

Biology 05LA Fall Quarter 2013 Introduction

EXCUSED ABSENCE REQUEST FORM __________________ Please provide the information requested below.

Todays Date:

Student Name: Email address: Name of TA: Date of Missed Lab:

SID#:

86
Biol 05C

Course (circle one) Biol 05LA Biol 05B Lab Day: Lab Time: Date and Time received:

_______________ Briefly describe the reason for your absence.

If there is a possibility of your being rescheduled and you have a job, please give us your work schedule so that we do not schedule you to a lab time that conflicts with your work.

Once you have filled in the above, firmly attach any other materials (such as original copies of excuses and due assignments) to this form and place these documents in the collection box located on the door of the course academic coordinator.

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Biology 05LA Fall Quarter 2013 Introduction

Self-Scheduled Library Exercise: Finding Journal Articles in the Orbach Science Library
Library instructional sessions for Biology 5A, have been scheduled during the 3rd 7th weeks of the session October 15th through November 15th, 2013. Please make sure that you complete the Library Exercise because a portion of your final lab grade depends on your successful completion of this exercise.
The purpose of the exercise is to help you learn how to find scholarly articles in a university library. At the university and later in your scientific career, you will find that good research skills in the library are essential for success.

The Biology 5LA library exercise consists of two parts, which are to be completed in the following order: Part 1. An online tutorial on library research for biology. (You are to do this part on your own. You must complete this

tutorial to be admitted into the library session (Part 2)).


Part 2. A hands-on session in the Science Library finding journal articles using BIOSIS Database the Scotty catalog. (This part will be done during a scheduled session in the Library or online). Instructions for Part 1 1. Go to the Library Home page http://library.ucr.edu and click on Bio5A Tutorial and Sign-up under the Upcoming Workshops section. 2. Click on Bio5A Tutorial, and complete the online tutorial (It will take 20 to 30 minutes to go through the tutorial). You will need to complete this tutorial before you come to the library for Part 2. At the start of the library session, there will be a short quiz on material from this tutorial. Those not passing the quiz will not be admitted to the exercise, but may try again later, after reviewing the tutorial. Don't worry; you will be able to pass the quiz if you go through the entire tutorial, paying careful attention to the material that it covers.

Access the online Bio 5A Tutorial from the UCR Library Homepage: http://library.ucr.edu/?view=help/tutorials/biology/index.html Sign-up for your library session atany the end of the tutorial. The online tutorial is available on the Web through computer having Internet access with Web-browser capability. You can use one of the many computers on the UCR
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Biology 05LA Fall Quarter 2013 Introduction

Instructions for Part 2 1. Sign up for ONE library session at http://library.ucr.edu/workshops/. The schedule is below and some webinars may be added. Sign up early before all the sessions fill up. There are no make-up sessions. The library sessions are held in room 122 of the Science Library and last about 50 minutes. Each session holds a maximum of 30 students and fills up quickly. 2. Complete the exercise as early in the quarter as possible. 3. Arrive early at the Science Library. Each session starts exactly at the designated time. Latecomers will not be admitted. 4. If you miss the session for which you signed up, try to sign up for another session. 5. Instructions for individual library assignments will be given to you during your session in the library or during the Webinar. 6. Please email Julie Mason (jmason@ucr.edu) or call 827-2817 if you need to change your session or cannot attend a session you signed up for.

Biology 05LA Fall Quarter 2013 Introduction

Bio 5A Library Exercise Times Fall Quarter 2013 Sign-up at http://library.ucr.edu under Workshops Each session holds a maximum of 30 Students/Webinar sessions hold 50 students

October 2013
Thursday, October 31 Tuesday, October 15 12:40 1:40 p.m. 3:40 4:40 p.m. 30 Students 30 Students 11:10 a.m. - 12:10 p.m. 30 Students 3:40 4:40 p.m. 30 Students

Thursday, October 17 12:40 1:40 p.m. 2:10 3:10 p.m. Friday, October 18 12:10 1:10 p.m. 3:10 4:10 p.m. Tuesday, October 22 2:10 3:10 p.m. 5:10 6:10 p.m. 30 Students 30 Students 30 Students 30 Students 30 Students 30 Students

November 2013
Friday, November 1 11:10 12:10 p.m. 1:10 2:10 p.m. 30 Students 30 Students

Tuesday, November 5 3:40 4:40 p.m. 30 Students

Thursday, November 7 1:10 2:10 p.m. Friday, November 8 1:10 2:10 p.m. 3:10 4:10 p.m. 30 Students 30 Students 30 Students

Thursday, October 24 3:40 4:40 p.m. Friday, October 25 12:10 1:10 p.m. 3:10 4:10 p.m. Tuesday, October 29 11:10 a.m. - 12:10 p.m. 30 Students 5:10 6:10 p.m. 30 Students 30 Students 30 Students 30 Students

Tuesday, November 12 5:10 6:10 p.m. 30 Students

Friday, November 15 12:10 1:10 p.m. 30 Students

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Introduction

LABORATORY SAFETY
The laboratory exercises that follow are designed to facilitate the learning of many basic biological concepts and NOT to put the student at unnecessary risk of injury. Avoidance of laboratory injuries requires the participation of both the laboratory instructor and the student. The instructor's responsibility is to point out major points of risk for each exercise and to describe the procedures for avoiding accidents that could result in injury. The students responsibility is two-fold. First, the student must listen to their instructor's safety advisories and act accordingly during the lab period. Second, the student must realize that childish or immature behavior in the laboratory is a major cause of accidents and that this kind of behavior has no place in the laboratory. Following are a number of rules and general guidelines for a safe and injury-free experience in these labs. 1) Notify your instructor immediately if you have any medical conditions (such as pregnancy, allergies, diabetes, immunological problems) that may require special precautionary measures in the lab. 2) Keep personal belongings that are not of direct use in the lab off the top of the lab benches (these should not be placed in areas where they will impede traffic in the lab). 3) In case of fire, evacuate the room in an orderly manner and assemble outside the building in the designated area. 4) In case of earthquake, be aware that you should not try to exit the room while the ground is shaking. Your best response is to DUCK, COVER AND HOLD until the shaking stops-only then should you evacuate and meet in the designated area. 5) Do not eat, drink, smoke, or apply cosmetics in the lab. 6) Confine long hair, loose clothing, and dangling jewelry. 7) Wear safety glasses at all times when instructed to do so by your TA. 8) Wear shoes at all times in the lab. 9) Cover any cuts or scrapes with a sterile, waterproof bandage before attending lab. 10) Do not perform unauthorized experiments. 11) Report all spills and accidents (including animal bites) to your instructor immediately. 12) Never pipette by mouth. 13) Wash skin immediately and thoroughly if contaminated by chemicals or microorganisms and notify your lab instructor. 14) Safety with hot plates or open flames: a) Always assume that hot plates are hot. b) Never leave heat sources unattended. c) Be careful to avoid letting hot H20 baths boil dry. d) Use appropriate apparatus when handling hot glassware. e) Keep chemicals away from direct heat. f) Keep flammable liquids (alcohol, acetone, etc.) away from open flames. 15) Do not allow any liquid to come in contact with electrical cords. Handle electrical connectors with dry hands. Do not attempt to disconnect electrical equipment that crackles, snaps or smokes. 16) Always place used or waste materials in the area or container designated by your TA. 17) Do not pick up broken glassware with your hands. Also never dispose of broken glass or other sharp objects in the paper waste. 18) Always wear disposable gloves when advised to do so by your TA. 19) Leave the laboratory clean and organized for the next student. 20) Students will not handle compressed gasses/regulators. 21) The departmental Chemical Hygiene Plan (CHP) and the Injury and Illness Prevention Program (IIPP) manual are available in the labs and may be consulted for further safety information.

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Lab 1 page 1

LAB 1: INTRODUCTION TO LIGHT MICROSCOPY: THE COMPOUND MICROSCOPE: ITS CARE, COMPONENTS AND PRINCIPLES OF USE.
Over the past 20 years or so, the light microscope has become an extremely valuable tool in modern cell and molecular biology. In fact, a substantial part of the lecture content of Biology 5A that you will be hearing this quarter is derived from studies utilizing various forms of light microscopy. A feeling for the importance of light microscopy can be gained with a brief scan of your text book (Biology, Campbell et. al., 9th ed.). On page 113, the intracellular distribution of three classes of cytoskeletal elements is shown by the use of fluorescent tags. On page 110, the distribution of DNA within a mitochondrion is revealed by another fluorescence technique. The localization of telomeres on a chromosome is shown on page 319. And finally, the dynamics of the cytoskeleton as it participates in the process of mitosis is vividly demonstrated on pages 232 and 233. These few examples should convince you of the power of modern light microscopy. While you will not be using any of these techniques in Biol 05LA, it is very likely that you may later in your undergraduate studies or thereafter. It is the purpose of this laboratory to prepare you for this eventuality. The focus of this exercise will be in two areas. The first will be in the area of basic microscopy. Here you will be introduced to the parts of the microscope and how these parts interact to form an image. Then you will be introduced to some basic principles of light microscopy that are essential in all forms of microscopy. We will conclude by providing you with some basic guidelines Oculars (10x) for microscope use that have relevance to all Diopter ring forms of light microscopy. The second area of focus for this lab will be about how to handle the microscope in a manner that will Binocular eyepiece not damage it. Microscopes are expensive tube Revolving and obtaining the best possible results in nosepiece microscope studies requires that the instrument being used is in top working Objective lens order. Please take heed of the safety guide (1 of 4) Mechanical stage Arm lines that will be presented as you work assembly Mechanical stage through this exercise your success in assembly Condenser focus Biology 05LA and future studies will be Condenser enhanced if you do so. Course focus diaphragm lever I. MICROSCOPE COMPONENTS
Fine focus Stage motion controls Condenser Brightness control Illuminator

For Biol 05LA, the microscopes you will use will be placed on the lab benches by the lab prep. staff. Thus you will not need to move them at the start or the end of the lab period.

Base

The first thing you will need to do is to look at Fig. 1 and familiarize yourself with the names of the parts of the microscope; return to this diagram as often Figure 1. Parts of the compound light microscope; as necessary so that you can learn these The Nikon Alphaphot. names. If the microscope at your bench is not a Nikon Alphaphot, a labeled diagram appropriate for your microscope will be provided.

Biology 05LA Fall Quarter 2013

Lab 1 page 2

Structural elements. 1. Binocular eyepiece tube. This assembly holds the ocular lenses. It is designed to allow adjustment of the distance between the oculars and the focus of one of them. The eyepiece tube is held in position by a setscrew (not shown). This screw should remain tightened at all times and not
used for changing the position of the eyepiece tube.

2. Revolving Nose Piece. The objective lenses are threaded into this structure. The nosepiece rotates in a manner that allows objectives of different power to be clicked into position in the optical path. There are grooves on the edge of the nose piece that allow it to be rotated easily. Note: do
not change objective by pushing or pulling on the objective this could loosen the objectives and result in scope or slide damage.

3. Arm and Base. These are structural elements that support the optical system. The light source is built into the base. Off to the side of the base are the combined on/off switch and rheostat which controls brightness. 4. The stage and associated parts. Stage. The stage is the plane surface on which slides are placed for viewing. Focus controls. These controls raise and lower the stage and thus vary the distance between the specimen and the objective lens (this distance is referred to as the working distance for that objective). The specimen is focused in this manner. The larger knob that moves the stage greatly when rotated is the coarse focus. The smaller knob that moves the stage only slightly is the fine focus. The coarse focus should only be used when working with low power objectives.
Further, the stage should never be moved upward with the coarse focus control when looking through the microscope - doing so could damage the objective and/or slide. Such damage can be prevented by watching, from the side of the stage, to see that the slide will not touch objective when the stage is raised with the coarse focus.

The fine focus control has a limited range of movement. In practice, this means that you will have situations when you will not be able to use the fine focus control to sharpen the image adequately. To correct this, rotate the 10x objective into position and then rotate the fine focus knob to the middle of its range of travel. Next, very carefully use the coarse focus to sharpen the image (this will require only the slightest movement of the coarse focus knob). Once this is done, the fine focus control should be usable.

Condenser mounting bracket. This bracket holds the condenser assembly and can be moved up and down with the condenser focus control. Mechanical stage assembly. This mechanism functions to precisely move the slide from side to side and/or front to back. It is has two parts: 1. Movable slide clamp. This device is spring-loaded and grasps the slide by its edge. 2. Stage motion controls. These are 2 knobs (located one on top of the other) that project downward from the stage - one moves the slide from side to side and the other from front to back.

Biology 05LA Fall Quarter 2013

Lab 1 page 3

The Optical Components. The microscope you are using has four optical components: 1) the light source, 2) the condenser lens, 3) the objective lenses, and 4) the ocular lenses. 1. The light source. Light from the sub-stage bulb is diffused by a frosted glass surface located above the bulb. Light intensity is controlled by the power/brightness knob. You will find that the amount of illumination necessary for adequate viewing is dictated by the material to be examined and the magnification used. It must be kept in mind that excessive brightness will result in a
substantial loss of image contrast.

2. The condenser. The function of the condenser lens is to focus the maximum amount of light from the light source upon the specimen. Because this lens condenses a broad, dim circle of light into a smaller and brighter spot, it needs to be focused properly for best results. For our microscopes, the condenser is properly focused when the condenser assembly is near the uppermost range of adjustment. The function of the condenser can be demonstrated as follows: a. Plug in your microscope and turn on the illumination to an intermediate level. b. Move the condenser to its uppermost position with the condenser focus control and open the condenser diaphragm completely with its control lever. c. With the lowest power objective in position, lay flat a small (2 x 2 or so) piece of paper over the hole in the center of the stage. When looking directly at the paper (not through the scope), you should see a bright spot of light projected upon the paper. d. Observe what happens to the spot of light when the condenser is lowered and then raised again. e. At this time you should clearly understand the value of having the condenser properly focused, that is, positioned near the top of range of movement. NOTE: At one level of focus,
the clear background of the microscopic field will have a grainy and colorful appearance. This grain can be eliminated by slightly lowering the condenser with the condenser focus control.

While a properly-focused condenser contributes to a high quality image, it can also cause a potential problem when viewing living specimens. This problem is that the small spot of illumination created by the condenser is also hot in addition to being bright. This heat may harm living specimens. To avoid such problems, you should make the required observations quickly, or lower the light intensity with the brightness control knob (do not lower the condenser). The condenser diaphragm regulates the diameter of the light beam passing through the condenser. As the condenser diaphragm is closed, specimen contrast increases. This phenomenon can be utilized to great advantage when observing specimens that have low inherent contrast such as living cells. You will have a chance to see this later. Be advised that microscopic resolution (see next section) is lost as the condenser diaphragm is closed. 3. The objective lenses. The function of the objective lens is to accomplish the first stage of magnification in the compound microscope. These lenses are threaded into the revolving nosepiece. NOTE: Remember that rotating the nosepiece should be done with the use of the
grooved surface on the edge of the revolving nosepiece and not by pushing or tugging on the objectives themselves. Doing so could loosen the objectives and eliminate the parfocality of the microscope this could result in scope or slide damage. A further word of caution about the objective lenses concerns lens cleaning - improper cleaning of the objectives can result in a scratched lens. This kind of damage is prevented by keeping in mind that all

Biology 05LA Fall Quarter 2013

Lab 1 page 4

exposed lens surfaces should be cleaned with lens paper only. DO NOT USE KLEENEX, LAB TISSUE, OR HANDKERCHIEFS! Your TA will demonstrate the best way to clean the lenses of your microscope.

At this point you should note the markings on the three or four objectives on the nosepiece. These are shown in Figure 2. Two points need to be made here: First, the magnification listed gives the magnification achieved only with the objective lenses. Since the ocular lens, to be discussed later, magnifies the image formed by the objective lens ten times, the value for each objective lens magnification times the 10x magnification contributed by the ocular gives the total magnification for the combined (compound) lens system. Figure 2. Objectives of the Nikon Alphaphot. The Second, numerical aperture (NA) is a measure of the light gathering capacity of the objective lens. The greater the value of NA, the greater the resolving power of the lens.
upper large numbers indicate the magnifying power of the objective; 4 - 100x. The number to the side or just below the mag. value indicates the numerical aperture of the objective; 0.1-1.25. The numbers at the bottom gives other information about the objective.

Some optical principles involving the objective lens system. The resolving power of a lens system is defined as the minimum distance that two points can be spatially separated and still be seen as two individual points. The resolving power of the unaided human eye is 0.1 mm (100 m). That is, if two points are spaced closer than 100 m, the human eye sees them as a single point. The maximum resolving power of the light microscope is about 0.2 m. Since many cellular organelles are 1.0 m or less in length, they are at the limit of the resolving power of the light microscope. Depth of field for a given objective is a term that relates to the thickness of the microscopic field that is in usable focus at a particular time. This definition may be obscure at the moment, but the following exercise will help you to understand it. Before you begin, please refer to the content of #I. in the SUMMARY OF MICROSCOPE USE PROCEDURES on page 9. You will be expected to use the techniques described there for this and all subsequent microscopy exercises. a. Obtain a slide with three overlapping threads of different color. b. Using the protocol referred to above, obtain a low power (40x) image of the area where the threads overlap and then determine which of the three threads is on top, in the middle, and on the bottom. c. Now focus as best you can on the middle thread. Can you still see the top and bottom threads clearly? d. While still focused on the middle thread, bring the medium power objective into position and sharpen the focus on the middle thread. Can you still see clearly the top and bottom threads? e. Repeat step d using the high power objective. Are the top and bottom threads still visible? You should have seen that depth of field decreases as magnification increases. This relationship can be used by the microscopists in two ways: First, lenses with a thicker depth of field should be used when either locating smaller specimens or when you want to study the spatial

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relationships of larger structures where high resolution is not important. Lenses useful here are low power lenses. Second, lenses with a thinner depth of field should be used when you want to study the spatial relationships of smaller structures where high resolution is more important. High power lenses with higher numerical apertures are most useful here. Parfocality of the objective lenses. A microscope is said to be parfocal if the object remains in focus (or close to focus) as the nosepiece is rotated to bring different objective systems into position. Are our microscopes parfocal? This is determined as follows: a. Using the techniques described in section #II in the SUMMARY OF MICROSCOPE USE PROCEDURES, place a slide with the letter "e" on the stage of your microscope and obtain a sharply focused image of the "e". Center the e in the field of view. b. Using the proper technique, rotate the nosepiece to bring the medium power objective into alignment. Does the letter "e" remain in focus (or close to focus)? Before going to step c, sharpen the focus and recenter the e in the field of view. c. Repeat above step with the high power objective. Does the letter "e" remain in focus? (Do not
change objectives or adjust the focus at this time there is another observation that you will need to make with this slide!)

If the answers to questions b & c are "yes", the microscope is parfocal. Why is parfocality important to the microscopist? The answer is simple and twofold: First, it makes specimens easier to find at high magnification. This is true because a parfocal lens system allows one to find the specimen at low power, and then maintain this focus when a higher power objective is selected. Second, it prevents damage to microscopes and slides. When prefocused higher power objectives are rotated into position, there is little chance of crashing objective into slide. Working Distance of the objectives lenses. As mentioned previously, working distance for a focused objective is the distance between the tip of that objective and the specimen being viewed. Working distance varies greatly between the objectives supplied with our microscopes. To observe this variability, do the following. a. Starting with the microscope configuration achieved in part c of the Parfocality exercise (40x objective focused upon the letter e) observe the working distance of the 40x objective. b. Rotate the 10x objective into position, focus, and note the working distance. c. Finish by rotating the 4x objective into position, focus, and note the working distance. Here you should see that working distance decreases with an increase in the power of the objective. While an awareness of the working distance for the different objectives has no direct influence upon the quality of the image, it does have two practical uses. First, the microscopist must always be mindful of the short working distances of high power objectives and the potential for unintended contact between the objective and the slide. This potential problem is averted by always beginning observations on a new slide with lower power objectives. Second, the long working distance of the low power (4x) objective offers plenty of room for exchanging slides and eliminates the need for lowering the stage when a new slide is selected.

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4. The Ocular Lenses. The ocular lenses use the real image formed by the objective lens as its object and produces the virtual image that the viewer sees (please see Figure. 3). The reason that the virtual image has this name is because it has the virtual appearance of being present in a plane located below the microscope. Once again, this type of microscope is called compound because the image that the viewer sees is formed by two lenses in Image optical series. Observations relevant to the virtual image of the compound microscope: Obtain a focused image of the letter e at low power. What direction does the image move if you move the slide toward you? Away from you? To the right? To the left? Is the image upright or inverted?
Lens and iris of the eye projected upon retina

Ocular Real image formed by the objective lens

The presence of two ocular lenses (one for each eye) provides an advantage to the microscopist as well as a disadvantage. The advantage is that, with a binocular viewing system, the microscopist has greater depth perception of the selected field of view. The disadvantage is that because of individual differences in vision, the oculars need to be adjusted to accommodate different users. These adjustments are made as follows: Adjustment of inter-ocular distance (I.O.D.). While looking through the microscope, the eyepieces are moved apart or together to accommodate the varying I.O.D.'s of different viewers.

Objective lens Orientation of specimen

Condenser lens Condenser diaphragm

Illuminator lens

Adjusting focus of the ocular lenses. Note that one of Illuminator the oculars has a fixed focus (right side) while the other bulb is adjustable with the diopter ring (left side). This permits the viewer to compensate for differences in the Figure 2 An image forming Figure 3 An formingray raypath path vision of each eye. Making this adjustment: With the in a Compound Light Microscope in a Compound Light Microscope left eye closed and the right eye open, focus the microscope with the lower focus controls until you see a sharp image. With the right eye closed and the left eye open focus with the diopter ring until a sharp the image is obtained. The microscope is now adjusted to compensate for the differences between eyes.

Some people have difficulty using binocular microscopes that is not related to either of the above adjustments. One explanation for this problem is that the microscope has been damaged by a negligent previous user thus making binocular viewing impossible. Another common problem here is that the viewer is holding his/her eyes too close to the oculars. The solution is to back your eyes off slightly from the oculars and redo the two adjustments described above. If you suffer from near- or far-sightedness, you should use the microscope without your glasses; it corrects for faulted vision. The microscope cannot correct for astigmatism.

* * *
[next page please]

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PRACTICAL MICROSCOPE USE AND THE CONCEPT OF IMAGE CONTRAST In this section, we will look at some plant and animal cells with the goals of practicing and refining your microscope skills as well as introducing another important microscopy concept; contrast. Contrast. You have learned that what can be seen in the light microscope is limited by the size of the object. Another limiting factor in microscopy concerns image contrast. Contrast relates to the ability of the viewer to distinguish an object from its background. For example, it is easier to see dark spots on a white suit than it is to see dark spots on a dark suit. Problems of inadequate contrast are common in microscopy. In Biology 5 we will use two methods to increase contrast: partial closure of the condenser diaphragm and differential staining. 1. Closure of the condenser diaphragm. For each of the specimens that will be provided, your first observation will be of fresh, untreated tissue. As you will discover, it is difficult to see much detail when the condenser diaphragm is wide open. However, if you slowly close the diaphragm while observing the specimen, you should see that more detail becomes visible as the diaphragm is closed. This improvement is realized only up to a certain point, after which contrast is not improved and resolving power is lost. Examine the effect of manipulating the condenser diaphragm for each of the available tissues and then make a sketch of the best images in your lab notebook. NOTE: The condenser must be properly focused to optimize the use of the condenser
diaphragm to increase contrast.

2. Differential staining. Objects seen in the compound microscope are discerned as a result of their interaction with light. Biological material frequently absorbs only a small amount of light and thus has poor contrast. Differential staining involves the use of stains (dyes) which bind to various components of the specimen to increase contrast. Stains usually bind specifically to particular substances in cells or tissues with a unique chemical make-up. Today, we will use three different stains with three different binding affinities: Acetocarmine. This is a natural stain that comes from an insect. This stain binds to chromatin and stains it red; thus it is useful for identifying nuclei. I2KI. This stain is a mix of iodine (I2) and potassium iodide (KI). I2KI works through a chemical reaction between the iodine and the starch granules of green plants. The resulting starch-iodine complex has a deep purple/blue color. Toluidine Blue. Unlike the preceding two stains, toluidine blue is a metachromatic stain when applied to biological material at an acid pH. This means that when the stain binds to different cellular components, the color produced may be different and dependent upon the chemical composition of that component. The above stains will be used in the following exercise. For each of the specified tissues, you will be drawing images of the specimens reacted with these stains in your lab notebooks and comparing these images with those drawn from the unstained material. While doing this, you should be thinking about what you think is uniquely colored with the different stains.

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The Tissues. Each student will do the following tissues observations. Onion epidermis. Your TA will show you how to obtain a small strip of onion epidermis that is one cell in thickness. Once you can do this with ease, proceed as follows: 1. Make a wet mount of a small epidermal strip as described in the section below entitled the Making of wet mounts. Here you should use tap water as the mounting liquid and float the strip on the drop with the torn surface against the water. Dont forget to add a cover slip. 2. Place the slide on the microscope stage. At low magnification, find an area with a minimal number of bubbles. Work your way up to a 400x image. 3. Manipulate the condenser diaphragm until you obtain an image that has the best contrast. 4. Sketch this image in your lab notebook. 5. Perfuse a drop of acetocarmine under the cover slip as described in the section below. The best staining will occur near the edge of the tissue. 6. Repeat steps 2 - 4 above. Potato slice. Here you will make a wet mount of a thin section (slice) of potato tissue. Your TA will give you some tips about how to make a section that is thin enough without hurting yourself. Once you can do this with ease, proceed as follows: 1. Make a wet mount of a small thin section of potato. 2. Proceed as described in steps 2 - 6 for the onion epidermis with the exception of substituting I2KI for the acetocarmine used in step 5. The thinnest edge of the tissue will be the best place for making your observations. Cheek epidermal cells. Here you will observe the epidermal cells lining the inside of your cheeks. Use the flat end of a toothpick to scrape the inside of your cheek to remove some of these cells. Gentle pressure is all that is needed. Proceed as follows: 1. Make a wet mount of your cheek cells using a drop of your saliva as the mounting liquid. 2. Proceed as described in steps 2 - 6 for the above tissues with the exception of substituting 0.1% toluidine blue for the stain. Be careful not to withdraw liquid from under the cover slip too quickly - you might sweep the cells from under the cover slip. This concludes the practical part of this laboratory session. Please go to item III in the SUMMARY OF MICROSCOPE USE PROCEDURES on how to properly conclude a microscopy session. When finished, you should write a short summary (a sentence or two should be sufficient) of your results for each of the four contrast enhancing techniques (that is one for the condenser manipulation and one each for the three stains). Then you should use your results to compare and contrast the structure and composition of the cells that were examined. ***

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SUMMARY OF MICROSCOPE USE PROCEDURES:


I. Obtaining the desired image with your microscope: 1. Starting checklist: a. Plug in the scope, turn on the sub-stage illuminator, and raise the brightness to an intermediate level. b. Check to see that the low power (4x) objective is in position. If it is not, use the edge of the revolving nosepiece to move it into position. c. Check to see that the condenser assembly is close to its uppermost possible position. If not, use the condenser focus control to move the condenser assembly upward. d. Check to see that the condenser diaphragm is not completely closed. 2. With a slide on the stage of the microscope, open the movable slide clamp by pushing it to the left and position the slide so that it can be held by the clamp. Remember that the slide clamp holds the slide by its edge. 3. Using the stage motion controls, move the slide so the object you wish to observe is positioned directly over the condenser. (If you cannot see the specimen, you can focus on the edge of the cover slip this will give you an adequate starting focus.) 4. While looking at the microscope stage from the side, carefully move the stage to its uppermost position with the coarse focus control. 5. While looking through the microscope, slowly lower the stage with the coarse focus control until an image of your specimen appears. Sharpen the focus with the fine focus control. 6. If higher magnification is required, center an important feature of your specimen in the field of view and sharpen the focus using the fine focus control. Then rotate the revolving nose piece so that the next strongest objective clicks into position. Some slight focusing will be necessary here to produce a sharp image of the specimen. 7. If still higher magnification is required, repeat step 6. II. Changing slides: 1. Rotate the low power (4x) objective into position. Because of the long working distance of this objective, THERE IS NO NEED TO LOWER THE STAGE WHEN CHANGING SLIDES. 2. Remove the slide from the grasp of the slide clamp. 3. Insert the new slide into the slide clamp and move the specimen into the light path. If the focus was not changed, the new specimen should be in close enough focus for beginning your work. III. Concluding the microscopy session: 1. Rotate the low power (4x) objective into position and remove the slide from the grasp of the slide clamp. DO NOT LOWER THE STAGE!! 2. Using the stage motion controls, center the stage mechanism over the stage. 3. Clean the ocular lenses and the 40x objective if necessary. 4. Turn off the illuminator.

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Two Useful Microscope Techniques


Making of wet mounts: A wet mount is a common form of specimen preparation for light microscopy that is used for the observation of a wide range of specimens. They are prepared as follows: 1. Place a single drop of water or other mounting liquid on the center of a microscope slide. 2. Place the sample upon the surface of or into the mounting liquid as specified. Grasp the opposite edges of a single cover slip between your thumb and forefinger. Be careful here, coverslips are quite thin and easily broken. 3. Touch the outer free edge of the cover slip to the slide surface at a point close to the drop. While holding the edge of the cover slip against the slide and slightly angled from the slide surface, move the slide toward the drop until they touch - the meniscus of the drop should grab the cover slip. 4. Slowly lower the cover slip onto the drop and release it from your grasp as the cover slip approaches the slide. Perfusion of stains under a cover slip: An easy way to apply aqueous stains to a slide that has been cover-slipped is as follows: 1. Apply a full drop of your stain to the slide surface at the edge of the cover slip - be careful not to get the stain on the top of the cover slip. 2. Place the edge of an absorbent material, such as a small piece of a paper towel, against the edge of the cover slip opposite to that which you applied the stain. This should pull some of the stain under the cover slip. 3. Blot off any excess stain.

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Learning Goals/Desired Outcomes for Lab 1


1. Be able to identify and properly name each of the microscope parts listed below and to describe the specific function of the parts shown in bold using appropriate terminology.

a. ocular lenses c. diopter ring e. binocular eyepiece tube g. arm i. stage k. movable slide clamp m. stage motion controls o. coarse focus control q. fine focus control

b. base d. brightness control f. light source h. revolving nosepiece j. objective lenses l. condenser lens n. condenser focus p. condenser mounting bracket r. condenser diaphragm and lever

2. Be able to routinely utilize all of the microscope use procedures described here. 3. Be able to safely clean the ocular and higher power objective lenses. 4. Be able to adjust the inter-ocular distance and the oculars (diopter ring) for comfortable viewing. 5. Be able to describe how the objective and ocular lenses interact to form the magnified image that the viewer sees. 6. Be able to describe the relationship between resolving power and numerical aperture. 7. Be able to describe what the condenser lens does and how an improperly focused condenser can influence both image quality and the usefulness of the condenser diaphragm. 8. Be able to define the following terms: resolving power, depth of field, working distance, parfocal (parfocality). 9. Be able to describe how the following microscope parameters change as one moves from the lowest power objective to the highest power objective: (1) resolving power, (2) depth of field, and (3) working distance. 10. Be able to define, in your own words, the term contrast. 11. Be able to define, in your own words, the term differential staining 12. Be able to give a positive and a negative aspect of using the condenser diaphragm to increase contrast. 13. Be able to give a positive and a negative aspect of using differential staining to increase contrast. 14. Be able to respond to any questions relating to the 15 Mistakes table presented on the next page.

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15 Mistakes Commonly Made by Beginning Microscopists and the Consequences of These Mistakes.
1. 2. MISTAKE Attempting to rotate the fine focus control beyond its limited range of movement. Moving the microscope stage upward with the coarse focus control while looking through the scope. Not positioning the low power objective in the light path before changing slides. Leaving the high power objective in the light path when returning scope to storage. CONSEQUENCES This can strip the gears that drive the focusing mechanism. This can result in hard contact between the objective and slide resulting in damage to either or both. This can result in damage to the high power objective. The high power objective could be damaged. The mechanical stage assembly could be damaged if the protruding portion is banged against the side of the storage cabinet. The objective lenses can become loose if handled in this manner resulting in a loss of the scopes parfocality. This can result in damage to the objective lens or slide. The microscope could be accidentally bumped against objects (or your lab mates) and damaged by this collision. The whole eyepiece tube could fall off the microscope and be badly damaged. This can result in damage to the mechanical stage assembly. This results in a loss of the friction that holds the stage at a selected focus. In this condition the microscope will not stay focused. Time will be wasted in finding the plane of the focus of the next specimen. This will make finding a desired object much more difficult and time consuming. Less light will be available for viewing the specimen. The usefulness condenser diaphragm for adjusting specimen contrast will be lowered.

3. 4.

5. Not centering the mechanical stage assembly before returning scope to storage. 6. Changing magnification by pushing or pulling on the objectives rather than by rotating the grooved edges of the revolving nosepiece. 7. Not carrying microscopes to and from the storage area with two hands and in an upright position. 8. Loosening the screw that secures the eyepiece tube onto the microscope arm. 9. Improper use of the stage clamp.

10. Lowering the stage when changing slides or at the end of a microscopy session. 11. Not beginning all observations with the low power objective. 12. Improper focus of condenser.

13. Attempting to view low contrast specimens with the light too bright and the condenser diaphragm The specimen will probably not be visible. too widely open. It will be difficult if not impossible to gain a three 14. Improper adjustment of the inter-ocular distance. dimensional image of the specimen. 15. Attempting to view specimen with eyes too close The specimen will be difficult if not impossible to to the oculars. see.

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LAB 2: SPECTROPHOTOMETRY AND QUANTITATIVE DATA ANALYSIS


This exercise will introduce the use of a spectrophotometer (or colorimeter) to measure a particular chemical property of many biological (and non-biological) materials. We will also learn to quantify spectrophotometric data, learn several "wet lab" techniques, and learn how to properly prepare scientific graphs. Be advised that you will be using all of these newly acquired skills in the labs of the weeks to come, so come to lab prepared to master these skills your potential success in these future labs will be greatly enhanced if you do so. Absorption Spectra All solutions that are colored to the human eye absorb electromagnetic radiation in the visible portion of the electromagnetic spectrum (Fig. 1). The color that we perceive represents those wavelengths of light that are not absorbed by the substance, or, conversely, represents those wavelengths that are transmitted. For example, if a solution is green, it absorbs in the blue and red portions of the spectrum. If it is red, we would expect it to absorb in the green and blue portions of the spectrum. Another way of saying this is that a solution transmits light of a color complementary to that which it absorbs (Fig. 1, color wheel). The absorption of certain wavelengths of electromagnetic radiation is as characteristic of a compound as is its molecular weight, solubility properties, melting point or any other intrinsic property. Thus, the absorption spectrum of a compound may be used to identify it. The fact that a compound, solution or substance is not colored does not mean that it does not absorb electromagnetic radiation. It merely means that it does not absorb visible light. However, it may absorb strongly in other regions of the spectrum. Water, for example, is colorless, but it absorbs far infrared light, a band of wavelengths to which the human eye is insensitive. The radiation that is absorbed by water is converted to heat, and the water becomes warm. Many other compounds of biological importance absorb in non-visible regions of the electromagnetic spectrum. Absorbance and transmittance are measured with either a colorimeter or a spectrophotometer. The instruments used in Biology 05LA are technically called colorimeters. A colorimeter is an instrument that selectively projects narrow bands of visible light upon a solution (see Fig. 2). A spectrophotometer does the same thing but also can utilize wavelengths in non-visible regions of the near ultraviolet and infrared. Both colorimeters and spectrophotometers function in the same manner. Light from a source
Comparable Wavelength Lengths: (in cm) UCR to Rubidoux Size of blue whale. McDonalds hamburger. Common Name

106 104 102 100 10-2 10-4 10-6


X-ray. Infra red. Visible spectrum. Ultra violet.
Red (650nm) Violet (400nm) Orange (600nm) Yellow (550nm)

Hertzian waves; radio, TV, and microwaves.

Size of an amoeba Red blood cell. AIDS virus H atom.

Blue Green (450nm) (500nm)

10-8 10-10
Gamma ray.

Electron

10-12 10-14
Cosmic ray.

Figure 1: The Electro magnetic Spectrum. The range of wavelengths of the EMS is shown. The visible spectrum represents a narrow band of the EMS. The approximate wavelengths of the colors in the visible spectrum are given in the color wheel. Colors that are complementary fall on opposite sides of the wheel.

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that emits light at the required wavelength is focused upon a monochromator by a lens-like collimator. The monochromator, either a prism or diffraction grating, separates the light into its component wavelengths. Once the light has been separated, the wavelength required for the analysis is selected from all the available wavelengths. This selection occurs when the operator adjusts a control on the front of the instrument which moves a slit ( selector) that allows only the selected wavelength to pass. This wavelength is then projected upon the sample. The light that is able to pass through the sample (i.e. that which is transmitted) is collected by a phototube that converts the light energy into an electrical signal that is proportional to the amount of light that it received. This signal is then transmitted to a processor that quantifies the amount of light received. The output from the processor then sends to results to an output display on the front of the instrument. The results may be reported as either percent transmittance (%T) or absorbance (A). It is important to realize that both of these values are related to the difference in the amount entering (Io) and leaving (I) the sample.
T =
Collimator

I I0

Output display.
(analog or digital)

or

%T =

I 100 I0

A = log

I0 I
Processor

Light Source

1 2 3 Monochromator
(prism or diffraction grating

I0

4
Cuvette (with sample)

Phototube

selector (slit)

Figure 2. Essential parts of a spectrophotometer.

In spectrophotometry and colorimetry it is necessary to zero the instrument at each wavelength to be measured with a tube known as a blank. This tube contains the same solvent or solution that was used to dissolve the compound that you wish to measure - less that compound. The blank is important because it may contain a component that absorbs light at the same wavelength as the particular compound to be measured. In which case, the absorbance of this component would be detected in the sample, in addition to that of the compound of interest. This would result in an inaccurate (higher) absorbance value for the sample you are measuring. The possible contribution of the solvent or solvent component to the total absorbance reading is eliminated by zero-ing the instrument with the blank before a measurement is made. This is done by adjusting the absorbance display to read zero with the blank tube in the instrument. Once this is done, the sample tube can be measured. Again, this process must be repeated for each different wavelength to be used. Specific instructions for this operation as they relate to one of the instruments used in Biology 5LA (the Spectronic 20 D+) follow:

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Zero-ing the Spectronic 20 D+: 1. If the machine is not already on, it can be turned on with a clockwise rotation of the knob on the left side of the lower front panel at least 15 minutes before use. 2. Set the desired wavelength with the knob on the right side of the horizontal surface. 3. Make sure that the filter selection lever, found in the lower left corner of the lower front panel, is in the correct position for the selected wavelength. 4. Set mode to transmittance with the mode button; the left-most button on the upper front panel. Verify your selection by observing the red light next to Transmittance on the display panel. 5. With no tube in the instrument, set ZERO transmittance using the knob on the left side of the lower front panel. (clockwise rotation increases value and counter clockwise rotation decreases value) HANDLE THIS KNOB WITH LIGHT FINGERTIP PRESSURE ONLY otherwise you might not feel the stop at the end of the limited range of rotation of this knob and thus damage the instrument.
6.

Insert the blanking tube into the tube chamber covered by the hinged lid on the left side of the horizontal surface. NOTE: Before inserting the blanking tube into the instrument, you should make
a small mark on the lip of the tube with an indelible marker. When the tube is inserted into the instrument, this mark should be always be positioned toward the front of the instrument. This will insure consistent readings each time the tube is inserted. This precaution should be taken for any tube that will be inserted into the instrument repeatedly.

7. Set mode to absorbance with the mode button used above. 8. Set absorbance to ZERO using the knob on the right side of the lower front panel. Once again
use only LIGHT FINGERTIP PRESSURE rotation is also limited by stops for this knob. Here, counter clockwise rotation increases value and clockwise rotation decreases value. NOTE: If the display shows a reading of 1.999, several turns in the CCW direction will be required to ZERO the instrument. The machine is now ready for insertion and measurement of the sample at the selected .

9. Steps 2 8 must be repeated for each wavelength to be measured. Remember that transmittance is set with no tube in the instrument and that the correct filter position range must be selected in accordance with the selected wavelength. 10. At the end of the session, remove the last tube measured from the instrument. If yours is the last lab of the day, turn the instrument off with counter clockwise rotation of the knob on the left side of the lower front panel. Otherwise, leave it on.

****
In the first of today's exercises, the absorption spectrum of potassium ferricyanide, K3Fe(CN)6, will be determined. The second of today's exercises will demonstrate that the absorption of light at a specific wavelength can be used for determining the concentration of a chemical in solution.
At this time, it is necessary to warn you that K3Fe(CN)6 is a POTENTIALLY HARMFUL SUBSTANCE IF SWALLOWED OR EXPOSED TO HOT, CONCENTRATED ACID In order to minimize the health risk of handling this substance, keep in mind the above warning o Do not mouth pipette; handle the solutions with care and wash your hands thoroughly after handling o Also, when you have completed your determination, dispose of your solutions in the manner described by your TA

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Making an absorption spectrum of potassium ferricyanide (work in groups of 4 students). 1) Obtain 5 ml of 0.75 mM potassium ferricyanide in a 10 ml disposable test tube. 2) Use 5 ml of de-ionized water as the blank to zero the spectrophotometer, and take readings of the K3Fe(CN)6 solution every 10 nm over the 380 nm to 500 nm range. (13 total readings) 3) Graph the absorbance as a function of wavelength. Do this now. Be certain that your graph shows the wavelength where absorbance is greatest (the max) and the absorbance value at that wavelength you will need this information later. Quantitative Spectrophotometry Measuring the amount of light that a sample absorbs allows the quantification of how much material is present in the sample. Although you may have had a slow and tedious time taking readings for an absorption spectrum, you gathered a large amount of data so quickly that it is already entered on a graph. As you will see, these absorption values can be easily converted to molar concentrations. The relationship between the amount of light absorbed by a solution, and the concentration of the material in the solution is determined with the use of Beer's Law. Beer's Law describes the relationship between the absorbance of a given solution and the following parameters: c - the concentration (in moles per liter) of the solution; l - the path length; this is the distance (in cm) that light must travel to pass completely through the sample. For our labs, a test tube with an inside diameter 1 cm is used giving an l value of 1 cm. E - the molar extinction coefficient; this is equal to the absorbance, at a specified wavelength, of a 1.0 M solution of a particular chemical measured with a path length of one centimeter. With reference to the above parameters, Beer's Law appears as follows: Absorbance (A) = Ecl. Reference texts that describe the properties of solutions of different molecules will often give the molar extinction coefficient (E) values, but only for chemicals that are colored, or absorb wavelengths that can be monitored by readily-available spectrophotometers. For example, the E value of pnitrophenol at 410 nm is 1.83 x 104 liters per mole x cm. An exercise in practical quantitative spectrophotometry. In order to familiarize you with the practice of quantitative spectrophotometry, your group will be supplied with a numbered vial containing a solution of potassium ferricyanide K3Fe(CN)6 at a concentration that is known only by your TA. Before you proceed, make sure you record the number of your sample in your lab notebook. The goal of this exercise is to use your knowledge of Beer's Law and the spectrophotometer to determine the concentration of K3Fe(CN)6 in your sample. GENERAL COMMENTS Before you begin this exercise, a few bits of information must be given. The first concerns the absorption spectrum of K3Fe(CN)6. As you should know, aqueous solutions of this compound have a simple absorption spectrum, composed of a single discrete peak that corresponds with the wavelength of light absorbed maximally by the solution. [This is the " max" for K3Fe(CN)6.] The first task in the completion of this exercise will be to produce an abbreviated absorption spectrum for K3Fe(CN)6 that shows the location of this peak and a usable absorption reading at the max. Producing an absorption spectrum that meets these criteria is complicated by the fact that our spectrophotometers can only read an absorbance of less than TWO! Thus, if the concentration of your sample is too high, you will not be able to read its absorbance! In which case, it will be necessary to dilute your sample in

Biology 05LA Fall Quarter 2013

Lab 2 page 5

order to read its absorbance. Because we are trying to accurately determine the concentration of a sample, the required dilutions must be made in precise way. Making accurate dilutions. A formula for making accurate dilutions is as follows: C1V1 = C2V2 where: C1 = the concentration of the solution to be diluted; V1 = the volume of C1 required for the dilution; C2 = the concentration of the solution after dilution; V2 = the final volume of the diluted solution. In order to show you how this works, let's pose the following problem: Suppose you have a 1.0 M solution of sucrose and you want to make 100 ml of 0.2 M sucrose. (Another way to state the problem would be to ask what volume (V1) of 1.0 M sucrose (C1) is needed to make 100 ml (V2) of 0.2 M sucrose (C2 )? The solution is easy: realize that V1 is the unknown value; plug in the above known values into the formula; and solve for V1. After doing so, you find that V1 = 0.02 liters or 20 ml. What do you do with this value? Measure out 20 ml of 1 M sucrose and then add 80 ml of water to make up the final desired volume, 100 ml of 0.2 M sucrose. Applying this formula to the problem of diluting the K3Fe(CN)6 requires two minor modifications since you do not have a numerical value for C1. This problem is solved by making C1 = 1 and C2 = to the desired degree of dilution that you wish to make (1/2, 1/5, 1/10, or whatever). Determination of the concentration of K3Fe(CN)6 in your sample. As stated earlier, the first step toward making this determination is to produce an abbreviated absorption spectrum for K3Fe(CN)6 that shows its max. In order to do this, proceed as follows: 1) Obtain your solution of unknown concentration from your TA and record its code number in your lab notebook. 2) Pipette 5.0 ml of your unknown into a test tube and measure absorbance at 10 nm intervals from 390 nm to 450 nm, using the instructions described previously. Use water as your blank. Record these readings in your lab notebook. 3) Should you find that your unknown is too concentrated (absorbance > 2), dilute your sample as described above in a systematic manner until you are able to obtain a spectrum with a max that has an absorbance of greater than 0.5 absorbance units. Once again, the solvent for the K3Fe(CN)6 is water. 4) Once you have obtained readings over the specified range of wavelengths that meet the criteria described above, graph the absorbances as a function of wavelength and identify the max. 5) Now, you are ready to calculate, using Beer's Law, the concentration of K3Fe(CN)6 in your sample. The value of "A" for your calculation is obtained from the graph that you just made at the max. The molar extinction coefficient for K3Fe(CN)6 at its max will be calculated using Beers Law and the data you collected earlier for the 0.75 mM K3Fe(CN)6 solution. 6) Calculation of the concentration of K3Fe(CN)6 in the original sample (the one that was given you by your TA) requires that you take into account the TOTAL dilution factor that was used. (For example, if you diluted your original sample by a factor of 1/5th and you use the A value obtained from this diluted sample for your Beers calculation, the resulting concentration will be 1/5th that of the undiluted sample. Thus you must multiply the result of your Beers calculation by 5 to get the concentration of the original sample.)

Biology 05LA Fall Quarter 2013

Lab 2 page 6

In your lab notebook, you make sure that you include your graphs for this exercise, as well as the following values: the absorbance value for the max of the unknown K3Fe(CN)6 solution that you measured. the factor by which you diluted the unknown sample. the value of E that you calculated. the code number for your unknown sample and the concentration that you calculated for it.
Please show all calculations, including those for your dilutions.

Learning Goals/Desired Outcomes for Lab 2


1. Be able to define the following terms: absorbance, transmittance, absorption spectrum, path length, molar extinction coefficient, and max. 2. Be able to describe the relationship between the color of a solution and the wavelength of light absorbed by that solution. 3. Be able to list the 7 major components of a spectrophotometer and to provide a simple description of the function of each. 4. Be able to demonstrate competence in the operation of the provided spectrophotometers. 5. Be able to explain why a blank is required for spectrophotometric studies and to describe how the blank for a particular study is chosen. 6. Be able to describe how the value of E (the molar extinction coefficient) is determined for a given solution. 7. Be able to use Beers law to calculate the concentration of an unknown solution. 8. Be able to use C1V1 = C2V2 to make dilutions. 9. Be able to present spectrophotometric data in an appropriately labeled graph.

Biology 05LA Fall Quarter 2013

Lab 3 page 1

LAB 3: DIFFUSION, OSMOSIS AND THE PERMEABILITY OF CELL MEMBRANES The movement of materials between cells and their external environment as well as within the cell is essential to life. For example, the raw materials for cellular metabolism must be imported and the metabolic waste products resulting from this metabolism must be exported. Within the cell, substrates for the cells enzymes must move into the active site of the enzyme and the soluble products of these reactions then distributed throughout the cell. Given the fundamental importance of this traffic, an understanding of the mechanisms whereby these materials move is essential. Since both the internal and external cellular environments are aqueous, the materials to be moved are dissolved in water and thus form a solution (remember that in an aqueous solution, water is called the solvent and the materials dissolved in the water are called solutes). Solute movement may be separated into two broad categories. One of these requires the direct output of energy by the cell and is referred to as active transport. Here, a solute is moved against a concentration gradient of that solute, that is, from an area of low solute concentration to an area of high solute concentration. Thus active transport allows cells to generate concentration gradients that support a diverse array of cellular processes. You will be hearing much more about active transport in lecture. The other category of solute movement does not involve a direct expenditure of energy and is called diffusion. Diffusion will be the primary focus of todays lab. Your text defines diffusion as: the spontaneous movement of a substance [solute] down its concentration gradient from a region where it is more concentrated to a region where it is less concentrated. This movement continues until the solute is evenly distributed throughout the available space. The driving force for diffusion comes from the activity of the water (solvent) molecules in the solution. Water molecules are not static. In reality, they are in constant motion that is driven by the heat energy of the system. Evidence for this can be seen at high magnification in the light microscope when viewing suspensions of small particles rapidly vibrating in a random manner. This motion, referred to as Brownian motion, is the result of randomly directed collisions between the water molecules and the suspended particles. Similar interactions occur between solvent and solute molecules. The random nature of these collisions and the probability of more frequent collisions occurring in areas where solutes are more concentrated work together to move the solute molecules into the available space, that is, down their concentration gradient. Thus, anything that influences the number of collisions between solvent and solute can, in turn influence the rate of diffusion. Relevant possibilities here include: the initial concentration of the solute and the amount of thermal energy available (at higher temperatures the thermal agitation of the water molecules is more rapid and forceful). Diffusion rate is also influenced by the mass of the solute; more massive molecules have a greater inertia and thus require a greater force to move. Thus far only the diffusion of solutes has been considered. Osmosis is an alternative version of diffusion that involves the movement of the solvent across a differentially permeable membrane separating two regions of different total solute concentration. A differentially permeable membrane is a barrier that allows the free passage of water molecules but does not allow the free passage of solute molecules. The region containing the lower total concentration of solutes is referred to as being hypotonic (relative to the other side) and the region containing the higher total solute concentration is referred to as being hypertonic (relative to the other side). Osmotic water movement occurs spontaneously from the hypotonic to the hypertonic side. Here, the driving force for osmotic water movement is the thermal motion of the water molecules as described above. The reason that movement is in the hypo- to hypertonic direction is that the total [chemical] potential energy of the

Biology 05LA Fall Quarter 2013

Lab 3 page 2

water molecules on the hypertonic side is diminished by the greater number of energy consuming interactions between solute and solvent on the hypertonic side. Thus, there is a potential energy gradient between the two sides; higher on the hypotonic side and lower on the hypertonic side. In this situation, water molecules will move down the energy gradient, that is, from the hypo- to the hypertonic side. FACTORS INFLUENCING DIFFUSION RATE In these experiments we will study the influence of three factors upon diffusion rate. This will be done by making comparisons between the distances moved by colored solutes through a gelatin medium. The gelatin to be used is called agar and is composed of mostly water. As such, the agar approximates an aqueous system, but is much easier to use for this kind of study. The basic plan is that drops different solutions will be added to small test tubes containing agar. The tubes will then be incubated under the specified conditions for at least one hour. Since the incubation time for each of the tubes will be the same, the distances traveled by the solutes will be proportional to the relative diffusion rates of the solutes. The solutes that will be used are: KMnO4 (potassium permanganate, mass = 158 grams/mole) and aniline blue (mass = 738 grams/mole). Table 1 lists the different solute concentrations and incubation temperatures that will be used. With some careful thought, you should be able to deduce which tube comparisons can be used to reveal the effects of varying molecular mass, solute concentration, and temperature upon diffusion rate. Procedure: 1. Each group of 4 students should obtain 6 agar-filled test tubes and label them 1 6. 2. With Table 1 as your guide, and with the use of a different Pasteur pipette for each solute, add 1 drop of each of the specified solutions to the proper tube. 3. When all 6 tubes are complete, replace the stoppers and incubate each tube at the condition specified for that tube. Record the time. 4. After at least one hour, use a ruler to measure the diffusion distances of the solutes (see Fig. 1) and record these measurements in your lab notebook. Diffusion rate = distance moved (mm) / time (min.) 5. Remove the stoppers from the tubes and place them in the designated recycling container. Dispose of the tubes in the designated waste container. Analysis. First, describe how these variations in molecular mass, solute concentration, and temperature influenced diffusion rate and then offer an explanation for these results based upon what you have learned about diffusion.
Tube #
1 2 3 4 5 6

Solute
0.1 M KMnO4 0.1 M KMnO4 0.1 M KMnO4 0.1 M An.Blue 0.02 M An.Blue 0.01 M An.Blue

Incubation
4o C room temp. 35o C room temp. room temp. room temp.

Table 1.

origin solute front

diffusion distance false solute front

clear agar

Figure 1. Measurement of diffusion distance. This diagram approximates the appearance of a sample tube after the allotted incubation time. Diffusion distance will be the distance between the origin at the top of the agar and the solute front. Ignore any false solute fronts that may appear because the solute moved between the agar and the wall of the tube.

Biology 05LA Fall Quarter 2013

Lab 3 page 3

OSMOSIS. Many cell types experience situations where the total solute concentration is different on the inside and the outside of the cell. When this occurs, water can move into or out of the cell depending upon the relative concentration of solutes on either side of the membrane. These water movements can have a significant influence upon cell volume or upon the level of hydrostatic pressure within the cell. In animals, osmotically driven water movement can drive many important secretory events such as sweating. However, in other animal cells, large changes in cell volume can be detrimental. In plants, osmotically driven water movement can contribute to a skeletal system that supports young plant parts or drive bulk solute flow through the plant. Given the diverse ways that these water movements influence both plants and animals, an understanding of the mechanism enabling this movement is essential to the biology student. This study will also introduce another expression of solute concentration called osmolarity. As you should know, a 1 molar solution of a polar molecule like glucose contains 6.02 x 1023 molecules of glucose per liter of solution. However, when 1 mole of a salt like NaCl is combined with water to make a 1 molar solution, the NaCl dissociates to give 1 mole of sodium ions (Na+) and 1 mole of chloride ions (Cl-). The dissociation thus doubles the total solute concentration of this solution. Consequently there are twice as many solute molecules in this solution that can deplete the total potential energy of the water molecules in the solution. As a result, the osmotic effect of a 1.0 M NaCl solution is twice that of a 1.0 M solution of a polar molecule. Osmolarity is an expression of concentration that accommodates this situation. Here, what needs to be understood is that the osmolarity of a solution is an expression of the total concentration of solutes expressed as molarity. For example, a 1.0 M solution of NaCl has an osmolarity of 2 osmolar and a 1.0 M solution of CaCl2 has an osmolarity of 3 osmolar. Given this information, it follows that osmotic water movement will occur in response to differences in osmolarity but not necessarily to differences in molarity. Modeling Osmosis: In this experiment, we will create artificial cells with the use of a synthetic differentially permeable membrane (called dialysis tubing). By filling these artificial cells (dialysis bags) with solutions of varying concentration and then placing them in beakers with solutions of varying concentration, we can model the osmotic challenges imposed upon real cells by changes in internal and external solute concentrations. Table 2 lists the 4 conditions that will be tested. Constructing these dialysis bags is fairly simple. Dry, precut strips of dialysis tubing are soaked in a beaker of distilled water. The strip is removed from the water and one end of the strip is tied tightly with a short piece of string. The other end is then opened in a manner comparable to opening the plastic bags in the produce section of the supermarket. Once opened, the desired solution is put into the bag. The bag is then closed by tying a knot with one end of a longer piece of string. A piece of colored tape is attached to the free end of the string and marked with the appropriate number. The bag is then weighed and placed in the specified incubation solution. After one hour, the bags are reweighed to determine if water has moved into or out of the bag. In this manner should see how differences in solute concentration on either side of a membrane affect the direction of osmotic water movement. Procedure: 1. Each table should obtain 2 400 ml beakers and then label each with either H2O or 0.5 M sucrose. The groups at each table will share these beakers. 2. Add 200 ml of the correct solution to the beakers.

Biology 05LA Fall Quarter 2013

Lab 3 page 4

3. Each lab group should obtain 4 strips of dialysis tubing and soak them in distilled water until they are soft and pliable. 4. With reference to Table 2, and using the method described above, create dialysis bags with the specified contents. 5. As each bag is finished, trim the excess string from each end of the bag (dont cut off the label). 6. After weighing and recording that value (in grams), place them into the specified bathing solution. 7. Incubate the bags for about an hour (no longer). At the end of that time, quickly blot the excess solution from the surface of each bag, weigh it, and record the weight.
Bag #
1 2 3 4

Bag Content
10.0 ml H2O 10.0 ml H2O 10.0 ml 0.5 M sucrose 5.0 ml 0.5 M sucrose + 5.0 ml H2O

Bathing Solutions
water 0.5 M sucrose water 0.5 M sucrose

Table 2

Analysis. For each trial, give the tonicity (either hypo-, iso-, or hypertonic) of the bag content relative to the bathing solution. After considering your results, what generalizations can you make about how differences in tonicity influence the direction of osmotic water movement? Osmosis In Living Plant And Animal Cells. The osmotic relationships between plant and animal cells with their environment are closely regulated in both cell types, but are not identical. Much of this difference is related to some fundamental differences between these cells. Plant cells differ from animal cells in many ways, but only two are important to this discussion. First, plant cells possess a fairly rigid cell wall. Second, healthy plant cells are most often hypertonic in comparison to their immediate exterior. This circumstance has a significant osmotic consequence. Given the hypertonic condition of cells interior, water should move into the cells by osmosis. If the cells were not bounded by the cell wall, water would keep moving into the cell until it burst. This does not happen. Instead, the cell wall resists the predicted osmotic swelling and pressure builds up within the cell. Plants use this turgor pressure to provide support for the plant or to drive long distance translocation of solutes. A characteristic feature of most animal cells is that they usually lack any sort of a structure comparable to the plant cell wall. Thus, the cytoplasm of the animal cell must be maintained in osmotic balance with the cells exterior. That is to say that the osmolarity of the inside and the outside of the cell should the same (isotonic). Maintaining this osmotic relationship is vital to the health of the cells because an animal cell exposed to a hypotonic environment would rapidly swell and eventually burst. Conversely, an animal cell exposed to a hypertonic environment would shrink because of excessive water loss. The following exercises will demonstrate the behavior of plant and animal cells exposed to different osmotic environments. In the first part of this exercise, we will manipulate the external solute concentration around the leaf cells of Elodea, and observe the consequences of this treatment with the light microscope. In the second part we will make similar observations on red blood cells (RBCs) exposed to hypo-. iso-, and hypertonic solutions. We will conclude by exposing rbcs to a range of glucose concentrations extending from hypo- to hypertonic in an attempt to estimate the osmolarity of the cells.

Biology 05LA Fall Quarter 2013

Lab 3 page 5

Osmosis in the Leaf cells of Elodea. Elodea is a common aquatic plant used in many fresh water aquaria. Its leaves are thin and comprised of fairly large cells. These features allow the microscopist to make useful observations of cell structure in intact leaves. Procedure: 1. With the use of a forceps, remove a healthy looking, green leaf from the tip of an Elodea twig. Make a wet mount of the leaf using water from the tank where it was collected. (Given this is
fresh water, there are very few solutes dissolved in it. We will consider the osmolarity of this solution to be hypotonic relative to the leaf cells).

2. Examine the leaf first with low power (40x total magnification). Then select a suitable cell and focus on it with higher power (100x total magnification no more!). Triangular cells that project from the edge of the leaf are most easily brought into clear focus, but if you focus carefully, cells the middle of the leaf can be studied. 3. Observe the following features in the healthy cell (it is important that you know what these cells look like in the healthy condition before applying the test solution): The cellulosic cell wall. The cell cytoplasm forms a layer just inside the cell wall. A large vacuole occupies the central core of the cell. This vacuole may be identified if you focus through the depth of the cell. It appears as a clear region in which no chloroplasts (globular green structures) come into clear focus. The chloroplasts are located in the thin layer of cytoplasm close to the cell wall. If the cell is healthy, the chloroplasts may be moving, but they always remain near the wall. The nucleus is often difficult to identify. It is found close to the cell wall and has a grey, shadowy appearance.

4. Once you have made the above observations, prepare another slide with a fairly large drop of 1.0 M NaCl. Then use a forceps to carefully transfer the Elodea leaf from your first slide to the drop of salt solution. Wait at least two minutes. 5. Apply a coverslip and blot off any excess salt solution. Observe the effect of this treatment upon the Elodea cells. Analysis. Describe the structural change in the Elodea cells elicited by the NaCl treatment. Explain the structural change that you observed. Osmosis in mammalian red blood cells. Red blood cells offer many advantages for this kind of study. They are readily available, easy to handle, and have a relatively simple structure characterized by a fixed size and unique shape (please see p. 911 of your text for a more detailed description of their structure). This latter feature will allow easy assessment of shape changes elicited by osmotic water movement. We will be using whole sheep blood obtained from a supply house. When handling these
cells, you must keep in mind that you are dealing with a cell suspension. Thus the cells will tend to settle with time. As a result, you will need to resuspend the cells anytime they are transferred or observed.

At this point, your group should obtain 5 ml of the RBC suspension* for your experiments. Your TA will dispense this into a clean test tube which you take to your lab station and place into a small beaker that is partially embedded in ice each table will have an ice bucket for this purpose. [*Your TA will prepare this mixture by combining 6.0 ml of whole sheep blood with 40.0 ml of isotonic saline.]

Biology 05LA Fall Quarter 2013

Lab 3 page 6

Part 1: Correlation of RBC structure in response to exposure to solutes of different tonicity. Procedure: 1. Obtain 3 small test tubes and label each with either hypo-, iso-, or hyper- (tonic). 2. In the proper tube, add 5.0 ml of hypo-, iso-, or hypertonic solution. 3. Once filled, add 0.2 ml of the RBC suspension to each of the 3 tubes -- keep in mind that you must re-suspend the RBCs by swirling the stock tube prior to dispensing. 4. Close each of these tubes with a small square of Parafilm and mix by gentle inversion. 5. Set these tubes aside for about 5 minutes before observation. 6. Students should now pair up to make the necessary microscopic observations. 7. For each of the samples, apply a drop of the RBC suspension to the center of a glass slide (remember to swirl before doing so), add a coverslip, and observe. [Caution, do not begin your
observations with the hypotonic sample.] Do not dispose of your sample tubes at this time; you will need to record their appearance (either clear or cloudy) in your lab notebooks.

8. Make careful drawings in your lab notebook of a few of the RBC's from each of the samples. Note the shapes of the cells and their relative numbers for each of the samples. Record this information in your lab notebook along with the appearance of the tube (either clear or cloudy) from which they came. Analysis. Describe the appearance the cells in each of the treatments. Explain the structural change that you observed. Part 2: Estimation of the osmolarity of red blood cells. An awareness of osmolarity is essential to biologists working with isolated cells and tissues because exposure of these isolates to anything but isotonic conditions would result in unintended swelling or shrinkage. Such changes would probably affect their experimental results. Thus, there must be some way to estimate the osmolarity of their experimental subjects. Actually there are several ways to do this, some requiring special instrumentation. We will use a relatively simple approach. In this exercise, a fixed amount of the RBC suspension will be exposed to a series of solute concentrations extending from hypo- to hypertonic. Somewhere near the interface of these extremes, the cells will be isotonic with their environment. At this point, the osmolarity of the cells and their environment will be the same. That is, the rate of osmotic water movement into the cells will equal that moving out of the cells. Your task will be to approximate the location of this point. By now you should realize that the cells exposed to hypotonic conditions will swell and lyse (burst) and those exposed to hypertonic conditions will be intact. Thus, we will be seeking to identify the concentration change that results in a change from the lysed to the intact condition (this concentration should be close to isotonic). Procedure: Two different solutes will be used for this study; glucose and NaCl. For each lab table, one group of 4 will use glucose and the other will use NaCl. Thus, each group will only need to prepare one set of dilutions as given in Table 3. When this study is complete, the two groups should share the data for the solute that they did not use. 1. Obtain 7 18x150 size test tubes and label each with one of the concentrations listed in the second column of Table 3.

Biology 05LA Fall Quarter 2013

Lab 3 page 7

2. Keeping in mind that you will be given a 0.5 M stock solution (C1) and that you will be making 10.0 ml (V2), of each concentration, you should now use C1V1 = C2V2 to solve for V1 (the volume of the 0.5M stock that will be needed to make the required dilution). Once calculated, record these values for V1 in the correct cells of Table 3. 3. Make the simple calculations needed to fill in column 4 of the table. 4. Make the required dilutions. Your TA will instruct you on how to do this efficiently. 5. Add 0.4 ml of the RBC suspension into each of the tubes (remember to re-suspend the cells before dispensing). 6. Close each tube with Parafilm, and mix by gentle inversion. Allow the samples to stand 5 minutes before collecting data.

Desired TONICITY molarity (C2) Hypotonic 0.0 0.1 0.15 0.20 0.25 Hypertonic 0.30 0.35

V1 (ml)

ml H2O to be added (V2 - V1)

Table 3. Dilution Work Sheet

Data collection. What you need to find here is the lowest solute concentration that contains intact cells. In order to do this you will need to go back to the tubes made in Part 1 of this exercise and ask two questions: which of these tubes contain intact cells and is there any apparent difference between the tubes with intact cells verses those with lysed cells? You should see that there is an easy way to tell the difference. Analysis. Using the visual feature alluded to above, what was the lowest glucose concentration at which intact cells were present? What was the lowest NaCl concentration at which intact cells were present? Explain this difference (remember that the values used are approximations).

Learning Goals/Desired Outcomes for Lab 3


1. Be able to define and differentiate between the following pairs or groups of terms: a) solute - solvent. b) diffusion - osmosis. c) hypotonic - isotonic - hypertonic. d) molarity - osmolarity. 2. Be able to define the following terms: a) concentration gradient. b) differentially permeable membrane. c) tonicity. 3. Be able to explain how variations in solute molecular mass, solute concentration, and temperature influence diffusion rate. 4. For an aqueous system where two solutions are separated by a differentially permeable membrane, you should be able to predict the direction of osmosis given the molar and/or osmolar solute concentrations on both sides of the membrane. 5. You should be able to explain why osmolarity differences are more significant than molarity differences when trying to predict the direction of osmosis. 6. You should be able to predict how plant cells will respond to exposure to a hypertonic environment and to explain the reasoning behind your prediction. 7. You should be able to predict how animal cells will respond to exposure to hyper- and hypotonic conditions and to explain the reasoning behind your predictions. 8. For the exercise where you estimated the osmolarity of some red blood cells, you should be able to explain why the lowest solute concentration at which the cells were unbroken represented a reasonable estimate of the osmolarity of the cells.

Biology 05LA Fall Quarter 2013

Lab 3 page 8

Biol 5LA Lab 3 In-class Worksheet


1. What is the driving force of diffusion?

Name __________________________

2. How does the molecular weight of a solute affect the rate at which it diffuses in a solvent?

3. If a solution of CaCl2 has an osmolarity of 6, what is its molarity?

4. Why are osmolarity differences more significant than molarity differences when trying to

predict the direction of osmosis?

5. In lab 1, why did you prepare the onion slide using water and the cheek cells using saliva? Use

the terms you have learned in lab today in your answer.

Bio 05LA Fall Quarter 2013

Lab 4A

Lab 4A: Genetic Testing Laboratory Detection of Alu Sequences by PCR *


It is estimated that there are 30,00050,000 individual genes in the human genome. The true power of PCR is the ability to target and make millions of copies of (or amplify) a specific piece of DNA (or gene) out of a complete genome. In this activity, you will amplify a region within your chromosome 16. Amplifying the Target Sequence The recipe for a PCR amplification of DNA contains a simple mixture of ingredients. To replicate a piece of DNA, the reaction mixture requires the following components: 1. DNA template containing the intact sequence of DNA to be amplified which in this case is genomic DNA that will be extracted from your cheek cells. 2. Individual deoxynucleotides (A, T, G, and C) raw material of DNA 3. DNA polymerase an enzyme that assembles the nucleotides into a new DNA chain 4. Magnesium ions a cofactor (catalyst) required by DNA polymerase to create the DNA chain 5. Oligonucleotide primers pieces of DNA complementary to the template that tell DNA polymerase exactly where to start making copies 6. Salt buffer provides the optimum ionic environment and pH for the PCR reaction The two DNA primers provided in this kit are designed to flank a DNA sequence within your genome and thus provide the exact start signal for the DNA polymerase to zero in on and begin synthesizing (replicating) copies of that target DNA. Taq DNA polymerase extends the annealed primers by reading the template strand and synthesizing the complementary sequence. In this way, Taq polymerase replicates the two template DNA strands. PCR amplification includes three main steps, a denaturation step, an annealing step, and an extension step (summarized in Figure 1). In denaturation, the reaction mixture is heated to 94C for 1 minute, which results in the melting or separation of the double-stranded DNA template into two single stranded molecules. The DNA templates must be separated before the polymerase can generate a new copy. The high temperature required to melt the DNA strands normally would destroy the activity of most enzymes, but Taq polymerase is stable and active at high temperature. During the annealing step, the oligonucleotide primers anneal to or find their complementary sequences on the two single-stranded template strands of DNA. In these annealed positions, they can act as primers for Taq DNA polymerase. Binding of the primers to their template sequences is also highly dependent on temperature. In this lab exercise, a 60C annealing temperature is optimum for primer binding. During the extension step, the job of Taq DNA polymerase is to add nucleotides (A, T, G, and C) one at a time to the primer to create a complimentary copy of the DNA template. During polymerization the reaction temperature is 72C, the temperature that produces optimal Taq polymerase activity. The three steps of denaturation, annealing, and extension form one cycle of PCR. A complete PCR amplification undergoes 40 cycles. The entire 40 cycle reaction is carried out in a test tube that has been placed into a thermal cycler. The thermal cycler contains an aluminum block that holds the samples and can be rapidly 1

Bio 05LA Fall Quarter 2013

Lab 4A

heated and cooled across broad temperature differences. The rapid heating and cooling of this thermal block is known as temperature cycling or thermal cycling.

5' 3'

3' 5'

Denature strands at 94C

5'

3' 5'

3'

Anneal primers at 60C


(Taq polymerase recognizes 3' ends of primers)

5'

3' 5' 3'

Primer

Taq polymerase

3'

5'

Primer
5'

3'

Extend at 72C
(Synthesize new strand)

5' 3' 5'

3'

5' 3'

3' 5'

Repeat cycle 40 times


Fig. 1. A complete cycle of PCR.

The Target Sequence The human genome contains small, repetitive DNA elements or sequences that have become randomly inserted into it over millions of years. One such repetitive element is called the Alu sequence. This is a DNA sequence about 300 base pairs long that is repeated almost 500,000 times throughout the human genome. The origin and function of these repeated sequences is not yet known. In this laboratory activity you will look at an Alu element in the PV92 region of chromosome 16. This particular Alu element is dimorphic, meaning that the element is present in some individuals and not others. Some people have the insert in one copy of chromosome 16 (one allele), others may have the insert in both copies of chromosome 16 (two alleles), while some may not have the insert on either copy of the chromosome (Figure 2). The primers in this kit are designed to bracket a sequence within the PV92 region that is 641 base pairs long if the intron does not contain the Alu insertion or 941 base pairs long if Alu is present. This increase in size is due to the 300 base pair sequence contributed by the Alu insert.

Bio 05LA Fall Quarter 2013

Lab 4A

When your PCR products are electrophoresed on an agarose gel, three distinct outcomes are possible. If both chromosomes contain Alu inserts, each amplified PCR product will be 941 base pairs long. On a gel they will migrate at the same speed so there will be one band that corresponds to 941 base pairs. If neither chromosome contains the insert, each amplified PCR product will be 641 base pairs and they will migrate as one band that corresponds to 641 base pairs. If there is an Alu insert on one chromosome but not the other, there will be one PCR product of 641 base pairs and one of 941 base pairs. The gel will reveal two bands for such a sample.

Fig. 2. The presence or absence of the Alu insert within the PV92 region of chromosome 16.

PV92
ALU

Genotype

DNA Size of PCR Products

ALU

Homozygous (+/+)

941 base pairs

Homozygous (/)

641 base pairs

ALU

Heterozygous (+/)

941 and 641 base pairs

(bp) 1,000 700 500 200 100

Fig. 3. Electrophoretic separation of DNA bands based on size. EZ Load DNA molecular mass ruler, which contains 1,000 bp, 700 bp, 500 bp, 200 bp, and 100 bp fragments (lane 1); two homozygous (+/+) individuals with 941 bp fragments (lanes 2, 6); three homozygous (/) individuals with 641 bp fragments (lanes 3, 5, and 8), and two heterozygous (+/) individuals with 941 and 641 bp fragments (lanes 4 and 7).

Bio 05LA Winter Quarter 2013

Lab 4A

Electrophoresis separates DNA fragments according to their relative sizes. DNA fragments are loaded into an agarose gel slab, which is placed into a chamber filled with a conductive buffer solution. A direct current is passed between wire electrodes at each end of the chamber. DNA fragments are negatively charged, and when placed in an electric field will be drawn toward the positive pole and repelled by the negative pole. The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than larger ones. Over a period of time, smaller fragments will travel farther than larger ones. Fragments of the same size stay together and migrate in what appears as a single band of DNA in the gel. In the sample gel above (Figure 3), PCR-amplified bands of 941 bp and 641 bp are separated based on their sizes. Lab Period 1 Isolation of Genomic DNA and PCR To obtain DNA for use in the polymerase chain reaction (PCR) you will extract the DNA from your own living cells. It is interesting to note that DNA can be also extracted from mummies and fossilized dinosaur bones. In this lab activity, you will isolate DNA from epithelial cells that line the inside of your cheek. To do this, you will rinse your mouth with a saline (salt) solution, and collect the cells using a centrifuge. You will then boil the cells to rupture them and release the DNA they contain. To obtain pure DNA for PCR, you will use the following procedure: The cheek cells are transferred to a microcentrifuge tube containing InstaGene matrix. This particulate matrix is made up of negatively charged, microscopic beads that chelate, or grab, metal ions out of solution. It traps metal ions, such as Mg2+which are required as catalysts or cofactors in enzymatic reactions. Your cheek cells will then be lysed, or ruptured, by heating to release all of their cellular constituents, including enzymes that were once contained in the cheek-cell lysosomes. Lysosomes are sacs in the cytoplasm that contain powerful enzymes, such as DNases, which are used by cells to digest the DNA of invading viruses. When you rupture the cells, these DNases can digest the released DNA. However, when the cells are lysed in the presence of the chelating beads, the cofactors are adsorbed and are not available to the enzymes. This virtually blocks enzymatic degradation of the extracted DNA so you can use it as the template in your PCR reaction. You will first suspend your isolated cheek cells in the InstaGene matrix and incubate them at 56C for 10 minutes. This preincubation step helps to soften plasma membranes and release clumps of cells from each other. The heat also inactivates enzymes, such as DNases, which can degrade the DNA template. After this 10 minute incubation period, place the cells in a boiling (100C) water bath for 5 minutes. Boiling ruptures the cells and releases DNA from their nuclei. You will use the extracted genomic DNA as the target template for PCR amplification.

Chromosome 16: PV92 PCR Informatics Kit Manual which is a product of Bio-Rad. Duplication of any part of this document is permitted for classroom use only

*The introductory information and protocol has been adapted from Biotechnology Explorer-

Bio 05LA Winter Quarter 2013

Lab 4A

Procedure
Lab Period 1 - Cheek Cell DNA Template Preparation
1. Obtain a cup containing saline solution from your instructor and go to your lab bench. Pour the saline into your mouth and rinse vigorously for 30 seconds. Expel the saline back into the cup.

2. Label the 1.5 ml micro test tube (a) on the bench with your seat number.

3. Transfer 1 ml of your saline rinse into the micro test tube (a) using a 1 ml pipet.

(a)

(b)

4.

Along with the rest of your classmates, spin your tube in a balanced centrifuge at full speed for 2 minutes. When the centrifuge has completely stopped, remove your tube. You should see a match-head sized pellet of whitish cells at the bottom of the tube. If you dont see a pellet of this size, decant the saline, refill your tube with more of your oral rinse, and repeat the spin.

(a)

Centrifuge

5. After pelleting your cells, pour off the saline. Being careful not to lose your pellet, blot your tube briefly on a paper towel or tissue. Its OK for a small amount of saline (< 50 l, about the same size as your pellet) to remain in the bottom of the tube.

6. Resuspend the pellet by vortexing or flicking the tube so that no clumps of cells remain. 7. Obtain a screw cap tube containing 200 l of InstaGene matrix from your TA. The tube number should match your seat number. Using a 1ml pipet, transfer all of your resuspended cells to the screwcap tube.

8. Screw the cap tightly on the tube. Shake or vortex to mix the tube contents.

Bio 05LA Winter Quarter 2013

Lab 4A

9. When all members of your team have collected their samples, place the tubes in the micro test-tube holder and incubate at 56C for 10 minutes in a water bath. At the halfway point (5 minutes), shake or vortex the tubes gently, then place back in the 56C water bath for the remaining 5 minutes.

Water bath 56C, 10 min Very Hot!

10. Remove the tubes, shake or vortex, and place the tubes in 100C dry bath. Incubate at 100C for 5 minutes. Be careful, the block is very hot!

Dry bath 100C, 5 min

11. Remove the tubes from the 100C dry bath and shake or vortex the contents to resuspend. Pellet the matrix by spinning at full speed for 5 minutes in a centrifuge.

Centrifuge
12. Place your screwcap tube in the TAs ice bucket. The TA will prepare your samples for PCR by transferring 20 l of your DNA sample to a PCR tube. The TA will then add 20 l of the master mix which contains the nucleotides, primers, magnesium ions, buffer and Taq Polymerase. The tubes are then placed in the thermal cycler for 40 cycles of amplification. Once the program has run, the PCR reactions will be frozen and stored until the next lab meeting.

Lab Period 2 Separation and Visualization of PCR Products


The PCR products you generated week 4 will be separated by gel electrophoresis. After thawing the PCR reactions your TA will add 10 l of loading dye. The TA will demonstrate the technique for loading samples into the wells of an agarose gel (A). The lid is then placed on the apparatus and the power cords are plugged into the power supply (B). Separation of the fragments will occur as the current flows through the gel. Note that the cords are color coordinated. The red cord represents the positive end of the field and the DNA will migrate toward that end of the gel (the dye front will be your visual indicator). After about 40 minutes at 120V the electrophoresis will be stopped and the gel transferred to a tray containing a concentrated staining solution (C). The DNA in the gel will bind the dye allowing visualization of the bands.

Can you determine whether you are homozygous (+/+), homozygous (-/-) or heterozygous? Genotype determined ____________________________________

Biology 05LA Fall Quarter 2013

Lab 4B page 1

LAB 4 -- HYPOTHESIS-BASED SCIENCE; AN INTRODUCTION TO THE HYPOTHETICO-DEDUCTIVE APPROACH TO PROBLEM SOLVING.


Science has been described as a way of knowing, that is, it is an activity whereby we learn about our world. This learning can be achieved by two different approaches. The first applies to inquiries in new or poorly understood areas and is referred to as discovery or descriptive science. Here, data is collected in the form of careful observations of a phenomenon of interest. This data is then carefully analyzed and recorded in a retrievable manner. As more and more data is collected, generalizations about this phenomenon can be made and our understanding thus increases. The type of reasoning by which this understanding was achieved is referred to as inductive because larger generalizations are derived from many smaller specific observations. In contrast, hypothesis-based science deals with questions in areas that are better understood. Here, problem solving begins with a well-defined question. The next step involves consideration of what is known about related phenomena and how we came to know these things. This information is then used as the basis for formulating a hypothesis, or possible answer to the question. The order in which this factual information is assembled is determined with the use of deductive reasoning. That is, it is ordered in a manner that places more general knowledge first and continues with information that is progressively more specific to the hypothesis being developed. There are two important concerns regarding hypothesis formation. First, hypotheses need to be falsifiable with an experimental test. Second, a workable experiment must be designed to make the test. Once a suitable hypothesis has been proposed and an experiment designed, a prediction is made about the expected outcome of the experiment given that the hypothesis is correct. This predictability is made possible by the deductive reasoning that was used in the formulation of the hypothesis in concert with an awareness of the experiment that will be performed. What is desirable here is an if/then relationship between the hypothesis and the prediction. This is presented as follows: if the proposed hypothesis is acceptable and the experiment is performed, then the predicted result should be attained. The experiment is then performed. If the prediction matched the experimental result, then the hypothesis is conditionally supported in light of the knowledge on which it was based. If the prediction did not match the experimental result, then there was a problem with the hypothesis, the experimental approach, or both. In either case, the hypothesis and/or the experiment should be reconsidered. In the real world of scientific research, it is not at all unusual for predictions not to match results in early efforts to answer a question. While this may seem discouraging to the new science student, the positive aspect of this approach is that it represents a learning process. That is, even with the failure of one iteration of the process, useful information has been gained for better focusing the questions, hypotheses, experiments, and predictions for the next round which should be more successful. In earlier editions of your text and this lab manual, the process described above is referred to as the Hypothetico-Deductive (H/D) approach to problem solving the origin of this terminology should be obvious from the above discussion. In Biology 5, we wish to demonstrate the practical value of the H/D approach by expecting the student to apply it to the investigative laboratories presented in this course. Some modifications of the procedure need to be made in order to make it applicable to work in the teaching lab. These are the subject of the following discussion. The H/D approach in Biology 5 laboratories. The major differences between the form of the H/D approach used by professional researchers and that which you will employ as beginning biology students are that the questions addressed by the lab exercises have already been defined as have the experimental protocols used for their study. These differences make the students job easier, but not trivial! Here is the amended procedure:

Biology 05LA Fall Quarter 2013

Lab 4B page 2

1) A fundamental demand for successfully applying the H/D approach is that the student must have attended all of the lectures associated with the lab exercise, read all of the notes taken from the lectures, and read all of the relevant assignments in the text. This requirement is directly comparable to the professional researchers need to have read all of the literature in scientific journals that pertains to their questions. 2) Next, it is time to carefully read (and reread as necessary) the exercise in the lab manual. You then must decide which experiments are associated with the different questions addressed by the lab exercise (these are found either at the end of this exercise or within the relevant lab exercise). 3) Now starts the task of formulating hypotheses (or proposed answers) for the questions. This process begins with the collection factual information relevant to the question. The sources for this information include your lecture notes, the text, and the lab write-ups in this lab manual. The challenge here is to organize this information deductively, that is moving in a progression from general to specific. This step is challenging; you should see your TA for help if you are having problems. Keep in mind that: a) hypotheses are possible answers/explanations. b) hypotheses reflect present knowledge about the subject area of the question. c) hypotheses should be expressed in terms that are testable by the experiments being done. That is, you should be able to make predictions of the outcome of your experiments given that your hypotheses are supportable. d) hypotheses can be eliminated but not confirmed with absolute certainty. 4) Next, you must come to understand the experimental strategy of the experiments you will be performing. In other words, you need to make sure that you understand the relationship between the data you will be collecting and the questions you are attempting to answer. In some cases, the relationship between your data and your hypotheses will be direct and straight forward. In others, some explanations are required to make this connection. This explanation is what we are calling experimental strategy. 5) At this point, the Bio 5 amendments end and the H/D process will continue as described above. Once again, predictions of the experimental results are made assuming that your hypotheses are supportable. Predictions are best presented in an if/then form; if my hypothesis is true and the experiment is done, then I should get this result. 6) Finally the experiment is performed and the result is compared with the prediction. If the result matches the prediction, then your hypothesis is supported. If the result differs from the prediction, you may need to reconsider your hypothesis, reevaluate your experimental technique, or both. A practical application of the H/D approach. The following is intended to help you see why this approach is so effective for problem solving and to give you a feeling for how we expect you to use this process for the presentation of your lab write-ups. This example from everyday life is derived from an experience which many of us have had. You get into your car, turn the key and nothing happens. Here, the question is obvious; why wont my car start? Before a possible answer (i.e. hypothesis) can be proposed, it is necessary to collect some information about how cars start because hypotheses need to be based upon available facts. To this end, you speak to your neighbor who teaches auto shop in the local high school. She tells you that the starter system works as follows: The key operates a switch that, when turned, directs electricity from the battery to the starter via a system of wires. This power does two things. First, it activates a solenoid which connects the starter motor to the engine. Second, it drives the starter motor which

Biology 05LA Fall Quarter 2013

Lab 4B page 3

actually turns the engine over and starts the engine. At this point you have plenty of information for proposing a few of the several possible hypotheses -- we will consider three of these. QUESTION: Why wont my car start?
Hypothesis
1. The battery is dead (i.e. it has no electrical charge).

Supporting fact(s)
Electricity from the battery is required to power the starter.

The experiment & Experimental strategy


The experiment: Attempt to turn on the lights. The experimental strategy: Since the battery powers most of the electrical systems in a car, a dead battery should also affect these systems (including the headlights).

Prediction
If the battery is dead, then the headlights wont work.

You now perform an experiment by attempting to turn on the lights. They come on. This result falsifies your hypothesis and you need to continue. However, this experiment was not a wasted effort. Now you know that electricity is available to run the starter.
Hypothesis
2. Electricity is not getting to the starter assembly.

Supporting fact(s)
Electricity is required for the starter to operate.

The experiment & Experimental strategy


The experiment: Connect a voltage meter to the power supply terminals on the starter. The experimental strategy. Since the experiment involves the direct measurement of electricity at the starter, the data that will be collected relates directly to the question.

Prediction
If electricity is not getting to the starter assembly, then the meter will not sense power at the starter when the key is turned.

The meter is connected to the starter and it shows that electricity is reaching it. This result falsifies your hypothesis and you need to continue. Once again, this experiment was not a wasted effort. Now you know that problem is probably with the starter assembly.
Hypothesis
3. The starter solenoid is not functioning.

Supporting fact(s)
The starter motor must be engaged with the engine for it to be able to start the engine. The solenoid engages the starter motor.

The experiment & Experimental strategy


The experiment: Replace the starter solenoid. The experimental strategy. Here the experimental data will relate directly to the question.

Prediction
If the starter solenoid is not functioning, then installation of a new one should allow the car to be started.

A new solenoid is installed and the car starts like new. Thus, your car would not start because the starter solenoid was defective. From this example you should see: The absolute necessity of learning as much as you can about the problem you are faced with before you attempt to formulate a hypothesis. The importance of presenting a hypotheses, supporting facts, experimental strategies, and predictions in the most concise manner possible -- This gives your problem solving effort the focus necessary to make it effective.

Biology 05LA Fall Quarter 2013

Lab 4B page 4

That an elaborate discussion of the experimental strategy is not required for experiments that provide data relating directly to the hypothesis. That the H/D approach is useful in the everyday world as well as in the laboratory.

Practical use of the H/D approach for the Write-Ups for investigative labs. Starting with the exercise on enzymes and continuing with the next lab on metabolism, you will be performing two truly experimental, investigative labs. As such they provide excellent opportunities for learning and appreciating the value of H/D approach as a process whereby we can expand our knowledge of biology. We consider this experience as one of the most important aspects of the curriculum of this course and your development as a science scholar! The guide that follows was prepared to help you with your weekly lab write-ups. The Rules: 1) While it is acceptable to discuss the lab write-ups with others, each student is expected to independently write his or her intros for submission and grading. 2) You must prepare the Introduction in advance of your weekly lab session. This is an essential step toward coming to lab adequately prepared.* A copy of your introduction (preparation guidelines follow) will be handed in to your TA at the beginning of the lab session on the day you are to perform the lab exercise. Late introductions will receive no credit. Also, you are reminded that you must perform the lab exercise to receive credit for its introduction. 3) Make sure that you understand what is expected of you before you begin. A major part of this exercise is to develop your ability to express yourself in a concise and focused manner. This takes time. 4) You are encouraged to have your TA preview and comment upon your introductions before they are handed in this is best done during your TAs office hours and not via email. 5) The introductions must be typed. 6) The results and discussion sections for these labs will be completed in the lab notebook. The processed data for the Enzyme (graphs and calculations) and Fermentation/Respiration (calculations) labs will be completed and handed in for grading at the times specified in the lab schedule.
* Adequate preparation also includes advanced preparation of any necessary data sheets for recording the data to be collected.

Preparation guidelines for the lab Introductions: The guide that follows provides specific guidelines for the preparation of your Introductions (and the Results and Discussion sections). The Introductions will be organized in the manner specified here or they will not be graded. The Introduction. For each of the questions listed for a particular lab exercise, you are expected to prepare separate introductory statements. Each of these should be organized as follows: Question #___: Here you should present the question you are addressing. 1) Your hypothesis: Insert here your hypothesis for the question. Keep in mind that:

Biology 05LA Fall Quarter 2013

Lab 4B page 5

a) You must learn as much as possible about what is known about related aspects of the question before you can formulate a hypothesis. b) Hypotheses should meet the criteria in item 3; a - d (page 2). c) Hypotheses should be expressed in the most concise and focused manner possible 2) Supporting argument for your hypothesis: Here you should write a concise, descriptive argument (entirely in your own words) in support of your hypothesis. This must be presented in a logical progression from general to specific - do not to assume that the reader is an expert in the field. You must also state the source of the facts used in your argument so that your TA can look these up. (e.g. Bio 05LA Lab Manual Lab # __, UCR, Summer 12 ed., pp.__-__, or Campbell Biology 9th ed. text, pp. __-__ ). Not citing the source of your facts will be considered plagiarism and will be penalized severely. Further, it is not acceptable to use direct quotes of factual information from the lab manual or the Campbell text in your argument. As stated above, this information must be presented in your own words. Two final comments: First, summary statements in the text or elsewhere are not facts and should be avoided. Second, because much misinformation is present on the WWW, citations from the internet will not be accepted. 3) Experimental strategy: What is needed here is a brief statement describing how the experimental approach to be used will relate to your hypothetical solution. What you need to convey here is the relationship between the experimental data that will be collected and the question you are attempting to answer. For example: a) How does the rate of color change in the reactions run in the enzyme lab relate to the activity of the alkaline phosphatase? b) How does a change in gas volume within the experimental tubes used in the fermentation and respiration experiments relate to the metabolic rate of the organisms in the tube? 4) Predictions: Remember that: predictions are best presented in an if/then form; if my hypothesis (here you need to actually state your hypothesis) is supportable and the experiment is done, then I should get the predicted result (here you need to actually state your prediction). Keep in mind that the prediction should relate to the actual experimental data that will be collected and not expressed in the more general terms used in your hypothesis. Completion of the write-up. The following items are required for the completion of the write-up. These should be presented in the lab notebook, but will not be graded. These items need to be prepared separately for each question. 1) Results. Begin by preparing the required graphs (if any) and then doing the required calculations. Next prepare a simple table of your processed data (e.g. the different rates for the different experimental conditions). Once the table is prepared, you then need to provide a brief statement describing what the table shows. Do not make interpretations of your results at this time. 2) Discussion. For each question you need to do the following. First, briefly and clearly state how well your predictions and results matched (be sure to mention the data that supports your claim). In the case of a good match, conclude with a simple declarative statement of what was learned this should be expressed in the terms of your hypothesis. In the case of a poor match, describe what you think went wrong and propose the specific changes that you think would improve your results were you to perform the experiment again. These changes may be to the manner in which you performed the experiment or to your original hypotheses. * * *

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Lab 4B page 6

Submission of introductions: Your introductions must be submitted in two formats. 1) The first is as a single computer file containing both questions uploaded through Safe Assign, a program designed to check your assignment for plagiarism. You will find the link on your TAs iLearn site under Course Materials. The assignment is due by midnight the night before your lab and you have only one chance to submit the file. Do not procrastinate! 2) The second is as a printed copy of the file. This is the version due in lab. You must submit both versions for full credit. The questions that will be addressed by the experimental labs this quarter are listed below or within the relevant lab exercise. The Enzyme Lab. 1) How will increasing substrate concentration affect the rate (v0) of an enzymatic reaction if the enzyme concentration is held constant for the different trials? 2) How will altering pH away from the optimal pH affect the rate (v0) an enzymatic reaction? The Fermentation and Respiration Lab these will be presented with the lab exercise.

Learning Goals/Desired Outcomes for Lab 4


1) Be able to define and differentiate between the following pairs of terms: a) inductive reasoning deductive reasoning. b) hypothesis prediction. (an educated guess will not be considered as an adequate definition of a hypothesis!) c) experimental protocol experimental strategy. 2) Be able to explain why gaining a thorough knowledge of the known facts related to a particular question is a vital prerequisite to proposing a hypothesis for that question.

Biology 05LA Fall Quarter 2013

Lab 4B page 7

Biol 5LA Lab 4 In-class Worksheet

Name __________________________

1. Write a hypothesis for each of the following questions: (1/2 point each) a. How will an increase in temperature influence the rate of diffusion of that solute down its concentration gradient? b. How will the molecular mass of a solute molecule influence the rate of diffusion of that solute down its concentration gradient? c. How will placing a cheek cell in a hypotonic environment affect the cell? d. If two solutions having differing osmolarities are separated by a differentially selective membrane, in which direction will the solvent flow?

2. Name three supporting facts you should include in the argument to support your hypothesis for question 1a. (1 point)

3. What would be the experimental strategy for question 1a? (1 point)

4. Write a prediction for the hypotheses you generated above for questions 1a and 1c. (1/2 point each)

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Lab 4B page 8

Some thoughts about the preparation of the two introductions you will be writing this quarter with particular emphasis on the Enzyme lab.
SUPPORTING FACTS. The biggest problem here is that many students make false assumptions about who is the audience for this document. They think they are writing this for their TA or the course faculty. This is definitely not the case. The correct target for this writing should be for someone who is not a scientist, but who is someone you want to enlighten about the topic. This means that you have to be very systematic about the way you develop the relevant scientific theory in support of your hypothesis and also to be careful to define all of the terminology used. In short, you want to prepare a document that any intelligent and curious person could pick up and understand. This requires that your argument be organized in a logical progression from general to specific. This can be challenging, but do-able for the science student. Start by carefully reading the questions so that you know what is being asked. Continue by reading the entire exercise in the lab manual. This will further clarify the question. Now that you have an idea about the question, it is time to learn about what is known about the subject of the question. To this end you must read your text, your lab manual, and your lecture notes for information pertaining to the question. When you come up with a piece of factual information that you think is relevant, write this down on a separate note card along with where you got it. Now you can FORMULATE A HYPOTHESIS based upon the facts you have collected this should not be too difficult for this lab. Hypotheses should be presented in the language of the question. You are now ready to assemble your argument for your hypothesis. This is where the note cards come in. These can be ordered in many different ways until you arrive at a progression that makes sense to you. At this time it can be seen what facts might be missing or what definitions might be necessary. These can then be inserted where necessary. The last bit of advice here is to put this aside for a while. When you come back to it, any remaining problems should be evident and easily corrected. Supporting Facts for Question 1 Define the term enzyme and each component of an enzymatic reaction. Define and describe the catalytic cycle of enzymatic reactions. Establish the active site as the part of an enzyme that binds substrate. Evaluate each step in the catalytic cycle to determine which step of the cycle is rate-limiting. Establish how the variable in question 1 could influence the rate limiting step determined above. Supporting Facts for Question 2. These will be much the same as for question 1. The difference lies in how the variable for question 2 influences the rate limiting step. Here you will probably need to broaden your fact search in the text. EXPERIMENTAL STRATEGY (ES) FOR BOTH QUESTIONS. Remember that ES is intended to make the connection between the actual data to be collected and your hypothesis. As stated in the lab manual, the key concern for the enzyme lab ES is: How does the rate of color change in the reactions run in the enzyme lab relate to the activity of the alkaline phosphatase? So, for this lab you will need to: Establish the basis for the color change. Describe how the color change will be monitored. Relate how the data collected is processed in order to come up with a value for reaction rate (v0)

While this sounds like a lot of work, the ES prepared for the first question is identical to that for the second question!!

Biology 05LA Fall Quarter 2013

Lab 5 page 1

LAB #5: ENZYMES Enzymes are organic catalysts that participate in the chemical conversion of one organic molecule to another. The molecule that is acted upon by an enzyme is known as its substrate and the molecule that is formed by the reaction is called its product. Enzymes participate in these reactions without being chemically changed as they are completed. Thus, one molecule of an enzyme could theoretically catalyze many conversions of substrate to product. This recycling of enzyme in multiple conversions of substrate to product is referred to as the catalytic cycle of an enzyme. It is described in the box to the right. When enzymes were first discovered, they were E + S ES EP E + P given a wide variety of names. For example, the enzyme that initiates the digestion of starch in the mouth, salivary The catalytic cycle of an enzyme occurs in 3 steps. amylase, was called ptyalin. Currently, enzymes are The enzyme and substrate bind to form an enzymenamed systematically by what they do. The ending "ase" substrate complex . While bound to the enzyme, the identifies a substance as an enzyme. This ending is substrate is converted to product resulting in an preceded by a stem which indicates a specific substrate, enzyme-product complex. The product is then released and the enzyme if free to bind another substrate molecule. the general nature of the substrate, or the type of action catalyzed by the enzyme. For example, succinic dehydrogenase removes two atoms of hydrogen from succinic acid, while hydrolases insert a molecule of water across a bond and thereby hydrolyze a compound. Most enzymes are proteins and are sensitive to environmental conditions. Enzymes, like most proteins, can be irreversibly damaged or denatured by high temperatures or pH extremes. There are a number of fascinating exceptions to these general rules: enzymes whose critical component is not protein, enzymes from organisms that thrive in super-heated ocean volcanic vents, and enzymes that operate quite well at very acidic or alkaline conditions normally viewed as deadly for all life. All enzymes, even the unusual ones, have conditions of temperature, pH, and concentrations of enzyme and substrates that promote their most efficient activity. In today's exercises, we will examine the activity of bovine intestinal alkaline phosphatase when exposed to variations in two of the above parameters. This enzyme normally operates in the intestines of cattle, away from the acidic stomachs (cattle have more than one), breaking down a variety of phosphorylated compounds found in their food. Alkaline phosphatase can hydrolyze the artificial substrate p-nitrophenylphosphate, giving us a convenient way to make quantitative measurements of its activity under different conditions. The basis for this convenience is derived from the fact that p-nitrophenylphosphate (pNPP) is a chromogenic substrate for alkaline phosphatase (and most other phosphatases). This means that an aqueous solution of pNPP, which is colorless, turns yellow when de-phosphorylated by alkaline phosphatase to form p-nitrophenol. This reaction is summarized as follows: alkaline
p-nitrophenylphosphate (colorless) + H2O phosphatase p-nitrophenol (yellow) + Pi (inorganic phosphate)

Further, the degree of yellowing resulting from the accumulation of the product (p-nitrophenol) can be measured with a spectrophotometer. A quantitative assessment of the activity of the enzyme is then achievable with the use of Beer's law. In this exercise we will conduct quantitative analyses of an enzyme out of its usual site of action (in vitro) so that we can manipulate substrate concentration and pH. In more advanced courses, you will learn how to extract and purify enzymes from biological material. Today, we will work with purified alkaline phosphatase that was purchased from a commercial source.

Biology 05LA Fall Quarter 2013

Lab 5 page 2

THE EXPERIMENTS: Reaction Time This trial is not a separate exercise. It is actually a part of each of the two exercises that follow. The reason it appears separately is to give you some practice performing this kind of experiment so that all of the remaining trials can be performed without error. Therefore, you should repeat it as many times as necessary until you are comfortable with the results that you obtained. You can then use the data collected here in each of the remaining exercises. Procedure: 1. Prepare a sample tube with 5 ml of high pH buffer to use as your blank. Use this to zero the spectrophotometer at 410 nm, and keep it for use at the beginning of each of the remaining exercises. 2. Prepare a second tube with 4.0 ml of substrate (stock concentration = 608 M). 3. Have a square of Parafilm ready. In the next step, you will want to add enzyme, top with Parafilm, mix gently by inverting the tube three times (do not mix violently), and get the tube into the spectrophotometer as rapidly as possible. 4. Add 1 ml of alkaline phosphatase and start timing immediately. (Make sure that you keep
your stock enzyme in an ice bucket!)

5. Take absorbance readings 30 seconds after you first added the enzyme. Then, take readings every 30 seconds, out to a total of 10 minutes (if necessary). Remember that you can repeat
this exercise if you think it is necessary.

6. Graph absorbance versus time for the data you collected.

****
Substrate Concentration In this exercise, we will examine the effect of varying substrate concentrations on the rate of the reaction. The substrate concentrations we will use have been prepared for you and are: 608 M (the stock dilution already done) 304 M 152 M 0 M (all high pH buffer) PLEASE NOTE THAT ALL OF THESE [SUBSTRATE]'S WILL GIVE A DIFFERENT REACTION RATE. Procedure: 1. Obtain 3 tubes and label each with one of the above substrate concentrations (remember, you have already done the 608 M sample). Add 4 ml of the appropriate substrate concentration to the proper tube and set aside. 2. Re-zero the spectrophotometer. 3. Beginning with the 304 M sample, proceed as described above in the reaction time trial. 4. Repeat this process for the remaining two samples. 5. Plot absorbance vs. time for each of the experimental trials on the same graph that you made for the 608 M run.

****

Biology 05LA Fall Quarter 2013

Lab 5 page 3

Effects of pH Alkaline phosphatase, as the name implies, is most active at an alkaline pH. Examine the effects of slightly acid pH by using the substrate pH 6.5 solution and the pH 6.5 enzyme. Procedure: 1. Re-zero spectrophotometer. 2. Obtain 4.0 ml of "substrate pH 6.5" in a clean tube (concentration 608 M) do not use the high pH substrate. 3. Have Parafilm square ready. Mix in 1.0 ml of low pH enzyme to the substrate tube, cap with Parafilm, mix by inversion, and take absorbance readings every 30 seconds out to a total of 5 minutes. 4. Plot absorbance vs. time for both the pH 6.5 and 10.4 (608 M) trials on the same axis. Remember that the Reaction time trial was run at pH 10.4.

Data Analysis: Because you will be responsible for the analysis the data from both of the experiments, it is important that you make sure that the raw data from both is recorded in your lab notebook prior to leaving the lab. Since the purpose of this laboratory is to evaluate the performance of alkaline phosphatase in a variety of experimental conditions, some parameter related to the performance of the enzyme must be used as a point of comparison for the different trials. We will use a value known as the initial velocity of the reaction for this purpose. This value, abbreviated v0, represents the time segment of the experiment when the reaction is proceeding at a constant and maximal rate for a given set of experimental conditions. The units we will use for v0 will be moles (of product) / unit time. There are many ways to calculate v0. The approach we will use is Absorbance vs. Time for Rxn. X based on the following:
2

Begin by plotting absorbance vs. time for each trial in a manner similar to that shown at right. (Note: when analyzing your data, you should plot all of the traces for a given experiment on the same graph.)

@ 410 nm

1.5 1

Once this is done, you should see that each trace 0.5 Y exhibits a relatively long segment that falls in a straight line (or very close to straight). For the graph above, this 0 0 50 100 150 200 250 300 linear segment falls between points Y and Z. The time (sec.) slope (A/t) of this portion of the trace is related to (but not equal to) the v0 for Rxn. X. The reason that this slope does not represent the initial velocity of the reaction is that A/t is a meaningless expression since absorbance is a unitless value. A step in the right direction is to use Beers law to convert the absorbances to concentrations of product. This conversion gives the change in concentration of product made vs. the change in time. However, this value does not tell us much more than A/t. So, it is necessary to change the concentration values to moles by multiplying the concentrations by the reaction volume used in the experiments: moles/liter (concentration) x .005 liter (reaction volume) = moles of product. Once this is done, you end up with moles/t which is an expression of initial velocity. QUESTION: If the product of Rxn. X is p - nitrophenol (E410 = 1.83 x 104 liters per mole x centimeter), what is the v0 of Rxn. X as plotted above? (ANSWER: 2.9 x 10-9 moles / sec.)

Biology 05LA Fall Quarter 2013

Lab 5 page 4

Learning Goals/Intended Outcomes for Lab 5


1. Be able to define the following terms: enzyme, substrate, product, enzyme-substrate complex, and enzyme-product complex. 2. Be able to use the above terms to describe the catalytic cycle of an enzyme. 3. Be able to define the term chromogenic substrate. 4. Be able to explain the experimental strategy for our investigations of the activity of bovine alkaline phosphatase. Your response should include: a. an explanation of why a chromogenic substrate and a spectrophotometer were used. b. a definition of the term initial velocity (v0). c. an annotated outline of the procedure used for converting the measured rates of color change in the reaction tubes to the v0s for these reactions. 5. Be able to explain the necessity of the 0.0 M substrate trial in the [substrate] experiment. 6. Be able to present and explain the supporting facts that you used to support your hypotheses for the two studies that you performed. 7. Be able to interpret the data presented in the following graphs by filling in the blanks in the table below with the number of the plot that is the best match for the listed description / explanation of the data. You must be able to explain your choice.
2 1. 2 2. 2 3.

Abs.

Abs.

Abs.

Time 4.

Time 5.

Time 6.

Abs.

Abs.

Abs.

Time

Time

Time

Description / Explanation The plot that depicts the slowest reaction rate The plot that depicts data for which a v0 cannot be validly calculated. The plot that shows the result of an experiment where the enzyme was denatured. The plot that depicts the fastest reaction rate. The plot that shows the result of an experiment where the substrate was contaminated with enzyme prior to the start of the experiment. The plot that shows the result of an experiment where the substrate was completely converted to product during the experiment.

Plot #

Biology 05LA Fall Quarter 2013

Lab 5 page 5

Biol 5LA Lab 5 Worksheet Name ____________________________ Lab partners: _______________________________________________________


1. Attach your hand-drawn graph for lab 5 to the worksheet. You should have plotted the absorbance versus time and have generated best fit lines for your data. The graph should also be properly labeled with a title and axis headings. (4 points) 2. Calculate the slope of each line and then convert these values to initial velocities (units moles/sec). Place values in the chart below. Attach calculations to worksheet and show all work for full credit. (4 points) Substrate 608 M pH 10.4 304 M pH 10.4 152 M pH 10.4 0 M pH 10.4 608 M pH 6.5 Slope vo

3. Discuss whether your results supported the two hypotheses you proposed in the introduction you turned in at the beginning of class. (2 points)

Biology 05LA Fall Quarter 2013

Lab 5 page 6

Reminder about Submission of introductions:


Your introductions must be submitted in two formats. 1) The first is as a single computer file containing both questions uploaded through Safe Assign, a program designed to check your assignment for plagiarism. You will find the link on your TAs iLearn site under Course Materials. The assignment is due by midnight the night before your lab and you have only one chance to submit the file. Do not procrastinate! 2) The second is as a printed copy of the file. This is the version due in lab. You must submit both versions for full credit.

Some guiding thoughts for the Fermentation and Respiration Lab:


Most students find preparing the Introduction for the Fermentation and Respiration lab somewhat more straight forward than that of the Enzyme Lab. This is largely because the material is a bit more familiar. The supporting facts for hypotheses regarding questions 1 & 2 can be derived from much of what you have learned so far in lab. Facts related to the Enzyme Lab and the Diffusion/Osmosis Lab are particularly relevant. However, these cannot be used without reference to how they apply to yeast cells. You should have no difficulty coming up with the necessary factual information in support of your hypotheses. Question 3 is much more challenging; here you need to think about how yeast cells and corn seedlings compare as organisms; thus, it should be obvious that you will need to search around a bit for relevant information. Points of comparison include: What are the differences between the composition of the food source for the two organisms? Are there differences between the food sources in the amount of biochemical processing that is required before the food can be used? What are the differences between the localization of the food source for each of the two organisms in comparison to where it is used? (very significant) What are the differences between fermentation and cellular respiration that might account for differences in the metabolic rate between the organisms (think about basic differences in biochemistry and the cellular compartmentalization between yeast and corn). The experimental strategies for these questions are definitely easier than for the preceding lab they are pretty well spelled out in the lab manual. Further, the ES for questions 1 and 2 are identical. That for question 3 is similar to that for the first two, but something must be added to make it relevant to measuring CO2 production in the seedlings. I hope this helps! Dr. Abbott

Biology 05LA Fall Quarter 2013

Lab 6 page 1

LAB #6: FERMENTATION AND RESPIRATION


All living organisms must continuously expend energy to maintain themselves and drive energy-requiring life processes. This energy is ultimately derived from the sun via photosynthesis. Because not all organisms are photosynthetic and the sun does not always shine, organisms need alternative sources of energy. Plants provide this energy in the form of organic molecules that contain large amounts of chemical potential energy. These molecules (e.g. sugars) are relatively stable and can be stored or transported. This energy is extracted by a stepwise, enzymatically-mediated oxidation of these molecules that results in the production of another energy-storing molecule, adenosine triphosphate (ATP). When the terminal phosphate of ATP is split off, the ATP is converted to ADP (adenosine diphosphate) and considerable energy is made available for cellular work. The ADP is then available for the regeneration of more ATP. Shown below are balanced summary equations for two classes of metabolism that provide most of the ATP that cells use.

C6H12O6 (glucose) + 2 ADP + 2 Pi

2 CH3CH2OH (ethanol) + 2 CO2+ 2 ATP + heat Alcoholic Fermentation

C6H12O6 + 6 O2 + 32 ADP + 32 Pi 6 CO2+ 6 H2O + 32 ATP + heat Cellular Respiration

The experiments that you will perform in this exercise are made possible by two features related to the above equations. The first is that gaseous reactants and/or products are characteristic of both metabolic processes. The second is that it is relatively easy to determine number of moles of a gas in a confined space with a device known as a CO2 gas sensor. This determination involves the measurement of the volume occupied by the gas of interest and then relating this volume to the number of moles of that gas. Another important feature of these equations is that they are in stoichiometric balance. This allows us to calculate the amount of any reactant or product if the amount of any other reactant or product is known. In our experiments, we will measure the rate of CO2 production in yeast cells undergoing alcoholic fermentation and the rate of CO2 production by corn seedlings undergoing cellular respiration. Once these rates are determined, the stoichiometric relationships of the reactions will be used to calculate the rates of ATP production for the organisms. The rate of ATP production is important because it closely approximates an organisms rate of energy utilization. This physiological parameter is termed metabolic rate. The reason that the rate of ATP production is so closely related to metabolic rate is because ATP is not stored (to any significant degree) by cells. Therefore, for it to be produced it must be recycled through the energy-releasing conversion to ADP + Pi. In other words, if ATP is being produced, it is also being broken down. ***** Note: The questions addressed in this lab are listed on p. 5 of this exercise. As was true for Lab 5, information relevant to this weeks questions comes from several different areas of the text. In addition to the obvious search words/terms, the following will be helpful: aerobic/anaerobic metabolism, angiosperm life cycle, and seed germination.

Biology 05LA Fall Quarter 2013

Lab 6 page 2

The experiments in today's exercise will be done in groups of four, each member of which should have the opportunity to set-up one experiment. Each student will be responsible for all data collected by the group. FERMENTATION Yeast cells can carry out both cellular respiration and fermentation although the fermentative pathway is preferred by most yeast under anaerobic conditions. The yeast being used in todays experiment, Saccharomyces cerevisiae, is unusual in that it will preferentially follow the fermentative pathway even when there is abundant oxygen to support the needs of cellular respiration. Because of this phenomenon (called the Crabtree effect) the amount of CO2 measured in this experiment will reliably reflect the CO2 being produced by fermentation. To measure the fermentation of glucose by yeast and the release of CO2 you will use a Vernier CO2 gas sensor inserted into an environmental chamber in which the yeast is fermenting. Since CO2 absorbs infrared radiation (IR) the gas sensor uses an LED to emit IR. A second internal sensor then determines how much of the IR is not absorbed by the CO2 in the environmental chamber. As the concentration of CO2 in the chamber increases, the amount of IR reaching the second sensor decreases. The CO2 gas sensor records the concentration of CO2 produced by the fermenting yeast in parts per million (ppm). Experimental protocol. A. Preparation of fermentation samples. 1. Set up the three fermentation samples in test tubes (17x150 mm) according to the values specified in the following table: Tube 1 2 3 Label 0.3 / 20% 0.6 / 20% 0.6 / 5% Vol. and Conc. of Sugar 6 ml of 20% glucose 6 ml of 20% glucose 6 ml of 5% glucose Amt. Yeast 0.3 g 0.6 g 0.6 g

2. Seal each of the above tubes with Parafilm and set them aside to activate. (During this time the yeast cells hydrate and the cells metabolic machinery becomes functional). During this period you should swirl the cells as demonstrated by your TA to speed the activation process. It is critical that the cells not be allowed to clump at the bottom of the tube during activation; what we are striving for here is to create a suspension of individual yeast cells. Also, be careful to avoid placing the tubes in an excessively cool spot such as the draft from an air conditioning duct. B. Measurement of CO2 evolution. The following steps will be carried out separately for each of the 3 samples/tubes. You are now ready to begin measuring gas production by your samples. At this point you need to determine whether or not your samples are ready to measure. Measurement can proceed when the sugar/yeast mixture begins to bubble and the Parafilm bulges upward.

Biology 05LA Fall Quarter 2013

Lab 6 page 3

1. Turn on Lab Quest recorder by pressing the silver button on the upper left-hand side of the unit. 2. Pour the entire yeast mixture from the test tube into a petri dish and place the dish inside the large environmental chamber. Attach the cover which should have one opening already plugged with a rubber stopper. Insert the CO2 sensor into the second opening. 3. On the touchscreen of the LabQuest recorder, tap the icon in the upper left corner that looks like a speedometer with the stylus. Next tap sensors and then data collection. Change the rate to 0.1 samples/s or the interval to 10s/sample. Change the length to 300s. Tap OK. To start the run tap the green arrow in the lower right. The graph will appear. Let the run continue undisturbed for 5 minutes. 4. After the 5 minute run, tap analyze, then curve fit and then check the box next to CO2. Tap the down arrow next to the choose fit box and select linear. Find the slope m which is ppm CO2/s and record this value in your notebook. 5. Repeat the measurement a second and third time if time permits. However, if the CO2 reading is getting close to 10,000 ppm after any trial perform the following steps: Remove the sensor, lid and yeast sample from the chamber. Then replace the CO2- laden air with fresh air from the room by waving the chamber around. Replace the yeast culture and reinsert the sensor. 6. When finished with each sample transfer the contents of the petri dish back into the original tubes and place them in the hot (60C) water bath for 15 minutes. Remove the tubes and allow them to cool before repeating steps 2-4 for all three samples. 7. See page 4 for a guide describing the calculations needed to process this data.

CELLULAR RESPIRATION Cellular respiration is the main metabolic pathway for ATP production that is used by animals, plants (especially at night), and many microorganisms. The conversion of sugars to CO2 and water liberates much more of the energy stored in the sugar than does fermentation (about 16 times as much ATP is generated in comparison to fermentation of the same amount of sugar). Measurement of the rate of CO2 production by an aerobic organism provides a useful measure of its rate of energy production. As for the yeast you will use a Vernier CO2 gas sensor inserted into a closed system containing corn seedlings undergoing cellular respiration. Cellular Respiration by Corn Seedlings Experimental Protocol: A. Preparation of corn seedlings: 1. Obtain and weigh a 250 ml Nalgene bottle and record its weight in your notebook. Tilt the bottle on its side and add about 25 corn seedlings (your TA will tell you how many) with radicles (the big white things that stick out of the corn kernel), ranging from 1.0 to 3.0 cm in length. Be careful not to handle them roughly or lump them at the bottom of the bottle they should be spread out in a single layer along the side of the bottle. This arrangement will allow all of the seeds to have adequate gas exchange with the air in the bottle. 2. Once the seeds are in the bottle, weigh the bottle with the seeds. The weight of the seeds can then be calculated and recorded on the worksheet. B. Measurement of CO2 production: 1. Insert the CO2 sensor into the neck of the Nalgene bottle.

Biology 05LA Fall Quarter 2013

Lab 6 page 4

2. On the touchscreen of the LabQuest recorder, tap the icon in the upper left corner that looks like a speedometer with the stylus. Next tap sensors and then data collection. Change the rate to 0.1 samples/s or the interval to 10s/sample. Change the length to 300s. Tap OK. To start the run tap the green arrow in the lower right. The graph will appear. Let the run continue undisturbed for 5 minutes. 3. After the 5 minute run, tap analyze, then curve fit and then check the box next to CO2. Tap the down arrow next to the choose fit box and select linear. Find the slope m which is ppm CO2/s and record in your notebook. 4. Repeat the measurement a second and third time if time permits. However, if the CO2 reading is getting close to 10,000 ppm after any trial perform the following steps: Remove the sensor then replace the CO2- laden air with fresh air from the room by waving the chamber around. Reinsert the sensor and continue with the trials. 5. When finished with the three readings transfer the contents of the bottle into a test tube and place it in the hot (60C) water bath for 15 minutes. Remove the tube and allow them to cool before transferring the seedlings back into the bottle and repeating steps 1-3. (This step may be eliminated at the discretion of the TA due to time constraints).

*****
1. Convert the slope from ppm/s to ml/hour: a. First convert ppm to ml using a proportion: average slope = ml of gas produced per second 106 volume of container* b. Second, convert seconds to hours: ml gas produced (from step 1a) sec x 3600 sec 1 hr = ml gas produced (use this value for part 2) hr

*The large chamber has a volume of 2,000 ml and the bottle has a volume of 310 ml

2. Determine how many moles of gas were produced: (1 mole of gas occupies 22.4 liters or 22,400 milliliters.) ml gas produced (from part 1) x 1 mole gas = hr 22,400 ml 3. Calculate rate of ATP production in moles.
FERMENTATION Remember that for every one mole of CO2 produced, there is one mole of ATP produced. So: moles CO2 prod.* x 1 mole ATP = moles ATP prod.** hr 1 mole CO2 hr
*(from step 3) ** (use this value for part 4)

moles gas produced (use this value for part 3) hr

CELLULAR RESPIRATION Remember that for every one mole of CO2 produced, there are 5.33 moles of ATP produced. So: moles CO2 prod.* x 5.33 moles of ATP = moles ATP prod.** hr 1 mole CO2 hr
*(from step 2) ** (use this value for part 4)

4. For comparisons between the yeast trials, the absolute rate of ATP production calculated in box #3 is sufficient. For comparisons between the yeast trials and the corn seedling results a mass specific value is required. Here the rate of ATP production (moles ATP / hour) must be expressed relative to the respective weights of the organisms measured. The mass specific rate is obtained by dividing the absolute rate of ATP production by the weight of the organisms giving: _____moles ATP____ gram organism x hour

Biology 05LA Fall Quarter 2013

Lab 6 page 5

Investigative questions addressed in this lab:


1. How will increasing the number of yeast cells affect the rate of ATP production by the cells (in moles ATP / hr) if the sugar concentration in the experiment is held constant? 2. How will decreasing the amount of glucose affect the rate of ATP production by the cells (in moles ATP / hr) if the amount of yeast in the experiment is held constant? 3. Will the yeast cells or the corn seedling have a higher metabolic rate when compared on a mass specific basis (in moles ATP / gram organism x hr)? [Here, it might be important to consider that we
are actually comparing fully hydrated seeds to dry yeast.]

Learning Goals/Intended Outcomes for Lab 6


1) Be able to define the term metabolic rate. 2) Be able to recite the balanced equations for fermentation and cellular respiration. 3) Be able to explain why measurements of the rate of metabolic gas production or consumption can be used as a valid measure of an organisms metabolic rate. Your explanation should include mention of the balanced summary equations for fermentation and cellular respiration. 4) Be able to explain why the use of balanced summary equations for metabolic studies of organisms with complex diets (consisting of many different food groups) is impractical. 5) Be able to explain why it was necessary to compare the metabolic rates of the yeast and the corn seedlings on a mass specific basis. 6) Be able to explain why it was necessary to include measurements of killed cells/seedlings in these studies. 7) Be able to present a factual and detailed explanation of why the yeast cells typically have a higher metabolic rate than the corn seedlings. 8) Be able to explain why it might be important to consider the fact that yeast cells have mitochondria when interpreting the results of experiments like the ones you performed. 9) Be able to explain why it was necessary to use corn seedlings having radicles only.

Biology 05LA Fall Quarter 2013

Lab 6 page 6

Biol 5LA Lab 5 Worksheet Name ___________________________________ Lab partners: _________________________________________________________


1. Perform the calculations outlined on page 4 of the lab and record the resulting answers in the table below. Attach calculations to worksheet and show all work for full credit. (4 points for table and 4 points for work)
Sample Slope in ml/sec Slope in ml/hr Moles gas produced /hr Moles ATP produced/hr Mass specific rate

Yeast Tube 1

Yeast Tube 2

Yeast Tube 3

Corn seedlings

2. Discuss whether your results supported the three hypotheses you proposed in the introduction you turned in at the beginning of class. (2 points)

Biology 05LA Fall Quarter 2013

Lab 7 Page 1

LAB 7: GENETIC TRANSFORMATION*


Genetic transformation is the act of altering the genotype of a cell by the introduction of DNA molecules from an external source. In nature, the source of DNA can simply be a virus or a DNA molecule that was released into the environment when a cell died and its DNA exposed. You probably have already heard about genetic transformation in lecture (i.e., Griffith's experiments involving the transformation of rough to smooth bacteria which lead to the discovery that nucleic acids were in fact the genetic material by Avery et al.) Today genetic transformation is used in the laboratory to engineer prokaryotes, animals, and plants for enhanced phenotypic characters and to repair deficiencies. The transformation process itself involves two distinct steps: 1. the entry of foreign DNA into the cell and 2. the expression of the genes residing on the foreign DNA molecule. In this experiment, you will be provided with bacterial plasmid DNA as a source of transforming DNA. Plasmids are very small, circular molecules that occur in nature. Plasmids replicate independently inside the bacterial cell. They have been manipulated by scientists for movement of genes in and out of bacteria in the laboratory. The plasmid we are using in today's experiment is called pUC118. It contains a gene that confers resistance to the antibiotic ampicillin. Bacteria that contain the plasmid can remain alive and grow in the presence of this antibiotic. Bacteria without the plasmid stop growing and may die in the presence of this drug. (Note: ampicillin is a derivative of penicillin).
pUC 118*

bla gene -lactamase

replication origin

* plasmid, University The objective of today's experiment is to ampicillin of CA, #118 genetically transform ampicillin-sensitive (or susceptible) resistance bacteria into antibiotic-resistant bacteria. The addition of a plasmid with an ampicillin-resistance gene will thus change the genotype and the phenotype of the bacterial cell.

Unlike the Stretptococcus (formerly Pneumococcus) studied by Griffith, Escherichia coli cells do not spontaneously take up DNA. Therefore prior to this laboratory, E. coli cells were treated with CaCl2 to enhance DNA uptake; these cells are competent for DNA uptake. Using the procedure described below, each group of students will mix DNA with the competent E. coli cells. Any cell that takes up the plasmid DNA and maintains it to express ampicillin-resistance is called a transformant. One third of the cells will be plated on ampicillin plates to determine the number of transformants. One third of the cells will be plated on Luria broth agar (LB) plates as controls. The third portion of cells will be serially diluted and plated on antibiotic-free media. The latter will be used to determine the total number of bacterial cells that were available for transformation. After growth, each lab group will score the number of transformants obtained and determine the total number cells available for transformation. These figures will be used to calculate transformation efficiency which, for our purposes, is the number of transformants obtained divided by the number of cells available for transformation.
*Note: There is a flow chart included on page 5 of this exercise to aid you in performing this experiment. Be sure to fill in each value, and time component prior to your laboratory period.

Biology 05LA Fall Quarter 2013

Lab 7 Page 2

To demonstrate bacterial transformation, each group of students will receive: 1. 2. 3. 4. 5. 6. 7. 10 drops bacterial cells (Escherichia coli) pUC118 plasmid DNA two ampicillin-containing petri plates (AMP, blue stripe label) five petri plates containing media without antibiotics (LB, unlabeled) sterile saline for bacterial cell dilutions three sterile 15-ml dilution tubes sterile L broth (bacterial growth medium)

Hazard: You will be using a plasmid DNA that carries an antibiotic resistance gene. While this poses no danger to you or your lab partners, it is important that we do not introduce the plasmid into nature. For this reason, we will be handling this DNA and the bacteria in accordance with Federal Guidelines for Recombinant DNA. It is critical that you dispose of all items that come in contact with DNA or bacteria in the specially designated containers. All items will be autoclaved prior to disposal.

Procedure: Be advised that accurate results in this experiment can only be obtained if the following two cautions are heeded: First, all measurements must be made as precisely as possible. Second, when transferring the prescribed volumes of the bacterial cell suspensions, care must be taken to make sure that the source container of cells is completely suspended before the transfer is made your TA will demonstrate the correct procedure for doing so. 1) Allow a microfuge tube containing 10 drops competent E. coli cells to thaw on ice. 2) Using a sterile long-tipped Pasteur pipette fitted firmly into a blue pipette pump (be careful here not to break the pipette by pushing too hard) slowly withdraw most of the volume of the thawed cells into the pipette. As you pull the pipette from the microfuge tube, briefly touch the tip of the pipette against the inner wall of the tube to clear any cell suspension from the outside of the pipette tip. Then, carefully transfer 3 drops (approx. 0.1 ml) of the cell suspension into each of two sterile microfuge tubes and close their caps. Return any cells still in the pipette to the tube of unused cells, cap the tube, and set it aside for later use. 3) Label one of the two tubes that you just filled "+", and the second tube "-". The "+" stands for plus plasmid DNA added; the "-" stands for no plasmid DNA added. 4) Your TA will add 1 drop pUC118 plasmid DNA to the "+" tube. (This is being done to ensure the accuracy of the DNA measurement.) Gently re-suspend the cells. 5) Add 1 drop sterile saline to the "-" tube. Gently re-suspend the cells. 6) Incubate both the "+" and "-" tubes on ice for at least 20 minutes. 7) During this incubation you should, get ready to determine the number of viable E. coli cells available for transformation. To do this, you will make serial dilutions starting with 3 drops of the remaining E. coli cells: a) Each group of students should collect four sterile culture tubes, and label them "A", "B", "C" and "D". b) To tube "A", add 9.9 ml of sterile saline. To tubes "B", "C" and "D", add 9.0 ml sterile saline. c) Set these tubes aside and continue the transformation experiment started earlier.

Biology 05LA Fall Quarter 2013

Lab 7 Page 3

8) Resuspend the cells in your "+" and "-" tubes and place the tubes into the 42oC water bath for 1 minutes*. Remove the tubes from the water bath promptly after this incubation.
* Be careful not to exceed this time!! Long periods at high temperature will kill E. coli. The o incubation at 42 C is called a heat shock: and makes the plasma membrane more fluid so that DNA can be taken up by the cells.

9) Using the pipetting technique described above, add 5 drops of sterile media (L broth) to both the "+" and "-" tubes and place at 37oC for 30 minutes. This is called the "out-growth" step and allows bacterial cells to metabolically recover. This period also allows time for the replication of the plasmid DNA and the transcription and translation of the antibiotic-resistance gene mRNA before challenging the cells with antibiotics. 10) During the "out-growth" period we will finish the dilution experiment started in Step 7: a) Using a new sterile pipette and the technique described in step #2 above, add 3 drops of the remaining unused E. coli cells to saline tube "A" and mix gently. b) Transfer 1 ml of the cell suspension from tube "A" to tube "B" and mix gently. c) Transfer 1 ml of the cell suspension from tube "B" to tube "C"; mix gently. d) Transfer 1 ml of the cell suspension from tube "C" to tube "D"; mix gently. 11) Label the bottom of three petri plates without antibiotics (no colored stripe) with "B", "C" and "D". Also, put the date and your group name on the bottom of the plate. This ensures that if the tops fall off (which they do occasionally), you will be able to recover your data. 12) Plate 3 drops of the cell suspensions "B", "C" and "D' on the appropriate plates. Your TA will demonstrate the plating procedure. Invert the plates and incubate overnight at 37oC. 13) We now return to the transformation experiment (Step 9). Obtain two ampicillin plates (AMP; blue stripe label) and two nutrient agar plates (LB; no stripe label). a) Label the bottom of one ampicillin plate with +AMP, the date, and your group name. Label the bottom of one LB plate with +LB, the date, and your group name. b) After the 30-min out-growth period, plate 3 drops of your "+" tube on the +AMP plate and another 3 drops on an + LB plate. c) Label the bottom of the second ampicillin plate with -AMP, the date, and your group name. Label the bottom of the second LB plate with -LB, the date, and your group name. d) Plate 3 drops of your "-" tube on the AMP plate and another 3 drops on the LB plate. e) Invert all plates, tape all of the plates together, and incubate overnight at 37oC. Your TA will oversee the length of the incubation period. SUMMARY: Each group should be incubating seven plates (total); four from the transformation experiment (+AMP, -AMP. + LB, and - LB) and three from the dilution experiment (A, B, and C). 14) After 16-20 hr at 37oC, each single growing bacterial cell will have divided thousands of times to give rise to a mound of bacterial cells which is called a bacterial colony. Your TA will show you a plate with E. coli colonies and will demonstrate a quick and relatively easy way to count them.
15) Counting will be done next week in lab. Carefully count and record the number of colonies on each plate. You should be able to provide answers to the following questions from your data.

Biology 05LA Fall Quarter 2013

Lab 7 Page 4

a. How many cells were available to be transformed in the "+" and "-" tubes? * b. What is the percent cells actually transformed? See the worksheet at end of the lab exercise for help here.

SPECIAL EXERCISE In each laboratory, two groups will undertake a modification of this experiment. These groups will be given the plasmid pBluescript which, like pUC118, is commercially available. The actual plasmid sample that will be provided will be a mix of two forms of this plasmid; one will be pBluescript while the second will be pBluescript into which a small piece of DNA has been cloned (added). pBluescript, like pUC118, contains an origin of replication, a gene encoding resistance to ampicillin, and the gene for one subunit of the lacZ gene (coding for the enzyme -galactosidase). galactosidase is comprised of 2 subunits (therefore it has a quaternary structure). This portion of the lacZ gene also contains a multiple cloning site (MCS) into which extra DNA can be cloned.

When pBluescript, lacking extra cloned DNA, is introduced into a host strain of E. coli, the gene carried by the plasmid can only produce only one portion of the enzyme -galactosidase and therefore cannot, on its own, cleave this sugar. However, if the bacterial cell genome contains the gene for encoding the other subunit of -galactosidase, a complete gene for the enzyme is available for transcription and translation of a fully functional enzyme. This raises the question as to how can we know that a complete, functional -galactosidase is present within the bacterial cell? This is actually fairly simple. Cells are grown on agar plates containing two extra components. The first of these is a chemical called IPTG which is an inducer for the enzyme (an inducer is part of the genes regulation mechanism) and the chromogenic substrate X-GAL. When X-GAL is acted upon by a functional galactosidase (X-GAL is very similar in structure to galactose) the resulting product is blue. Thus colonies of transformed cells are blue and easily recognized by color. But how can you know if DNA has been cloned into the MCS of the pBluescript plasmid? What must be kept in mind here is that the MCS is located within the coding sequence of the -galactosidase subunit gene. If DNA has been added in this location, the gene would be fatally interrupted and could not produce a functional enzyme subunit. Such plasmids, when introduced into E. coli, would not produce blue colonies when grown on media with IPTG and X-GAL. Instead, these colonies would be white.

Biology 05LA Fall Quarter 2013

Lab 7 Page 5

Procedure Follow the instructions for the lab for transforming and plating these plasmids. The only alteration is in the composition of the ampicillin plates. The two groups performing these modifications will use ampicillin plates that contain IPTG and X-GAL. These have already been added to the plates by the lab staff. Questions you should ask: 1. From the numbers of blue and white colonies on the ampicillin plate, what do you think the ratio pBluescript plasmid: pBluescript plasmid + insert were in the transformation mix? 2. Do you think the plasmids in the white colonies will be smaller or larger than the plasmids in the blue colonies? Why? **** The blue-white colony technology demonstrated here has proven very useful for isolating and copying genes from a wide variety of organisms with the use of the prokaryote E. coli. The utility of this technique for cloning genes is related to the very low frequency of being able to insert (ligate) a sequence of DNA (the gene you wish to copy) into the MCS present in the -galactosidase sequence. In practice, the frequency of insertion is 1 in 1000 or less. In should be evident that looking for colonies of cells containing the DNA of interest among the many not containing this DNA is literally like looking for a needle in a haystack. This selection is greatly simplified when all that needs to be done is look for the small number of white colonies that are present among the numerous blue colonies. As described above, the white colonies are comprised of cells unable to metabolize galactose and thus contain the inserted gene. Once this selection has been done, the cells of the white colonies are sub-cultured. The cloned gene can be extracted after the cells have multiplied to the number required.

Biology 05LA Fall Quarter 2013

Lab 7 Page 6

FLOW CHART
___ p dro s
s op r d _ __

10 drops competent E. coli cells

Remember - each time cells are dispensed or something is added, you must re-suspend the cells!! remaining competent E. coli cells ____ drops

+DNA

no DNA

___ drops cells __ ml

__ ml

__ ml

Ice for ___ min

42oC for ___min

Add ___ drops L broth ___oC for ___ min

A
___ml saline
__ drops

B
___ml saline
__ drops

___ml saline __ ml saline


__ drops
__drops

___ drops

___ drops

___ drops

___ drops

__drops

__drops

A
+ DNA *LB/AMP AMP + DNA LB LB
*LB/AMP

B
no antibiotic

C
LB antibiotic
no

D
no antibiotic

- DNA AMP

- DNA LB LB

no antibiotic

Incubate all plates For 16h @ ___oC

*For groups using pBS substitute LB/Amp/X-Gal plates for LB/AMP

Biology 05LA Fall Quarter 2013

Lab 7 Page 7

WORKSHEET FOR CALCULATION OF TRANSFORMATION EFFICIENCY:


1. Calculate the dilution factor for the transformants: Ask: How many transformed cells were generated from 3 drops of competent E. coli cells? 3 drops competent E. coli cells 1 drop plasmid +5 drops sterile media 9 drops of diluted cells (total) 3 drops of the diluted cells were plated. Therefore 1/3 of the diluted cells were evaluated on each antibiotic plate.

Dilution Factor =

# of drops plated on each plate 3 drops 1 = = total # of drops of diluted cells 9 drops 3

2. Calculate the number of transformed cells: Counting the number of colonies on each of the antibiotic plates gives you the number of transformed cells (Tdil) in 3 drops of your diluted cell mixture. Therefore the total number of transformants (Ttotal) = Tdil x dilution factor. Ttotal = Tdil x 3. Make this calculation for the +AMP plate. 3. Calculate the dilution factor used for determining the number of viable E. coli cells used in each transformation. Ask: How many total cells were the in 3 drops (= 0.1 ml) of competent cell suspension? There are two dilution factors to consider here. First, cells are diluted in the A, B, C and D tubes with saline (tube dilution factor). Second, only a portion of the cells in the B, C, and D tubes are plated on the petri plate for counting (plate dilution factor). Total dilution = Tube dilution x Plate dilution. Plate A B C D Tube Dilution Not plated 1 ml/10 ml = 1/10 1 ml/10 ml x 1/10 = 1/100 1 ml/10 ml x 1/100 = 1/1000 Plate Dilution 0.1 ml/10 ml = 1/100 0.1 ml/10 ml = 1/100 0.1 ml/10 ml = 1/100 0.1 ml/10 ml = 1/100 Total Dilution 1/100 = 10-2 1/1,000 = 10-3 1/10,000 = 10-4 1/100,000 = 10-5

Choose plate B, C or D and count the colonies. You want to choose the plate that has 25 - 250 colonies per plate. Some plates may not have enough colonies to count and others may have too many. Remember, 1 colony = 1 cell. 4. Calculate the # of cells available for transformation in each sample (3 drops of competent E. coli cells). Let B = # of cells on plate B; then # Cells per 3 drops = B x 103 Let C = # of cells on plate C; then # Cells per 3 drops = C x 104 Let D = # of cells on plate D; then # Cells per 3 drops = D x 105

5. Calculate the transformation efficiency.

Biology 05LA Fall Quarter 2013

Lab 7 Page 8

Ask: What fraction of the available cells were transformed? You know: # of transformed cells per 3 drops of competent cells (Step 2) # of viable E. coli cells per 3 drops of competent cells (Step 4) Therefore the transformation efficiency (TE) is: TE = # of transformed cells # of viable competent E. coli cells # of transformed cells per 3 drops # of cells per 3 drops

TE =

Learning Goals/Intended Outcomes:


1) Be able to define the term genetic transformation. 2) Be able to list the two steps in genetic transformation and to identify which step is associated with a change in genotype or a change in phenotype. 3) Be able to explain what plasmids are and how they are involved in genetic transformation. 4) Be able to explain why the E. coli cells used in our experiments are said to be competent. 5) Relative to the primary exercise on genetic transformation, you should be able to name the gene that we attempted to insert into the E. coli cells, name the protein that it codes for, and give the function of that protein. 6) Be able to explain how and why ampicillin was used in these experiments. 7) For each of the steps of the transformation protocol listed below, you should be able to explain how that step facilitated the transformation process. a) cold incubation after the addition of the plasmid b) the heat shock c) the outgrowth step (two considerations here) 8) At the end of the transformation exercise, we ended up with the 4 plates listed below. You should be able to explain what each of these plates contributed to our interpretation of the experiment. a) +DNA / AMP b) +DNA / LB c) -DNA / AMP d) -DNA / LB 9) Be able to define the term transformation efficiency. 10) Be able to explain how we knew whether or not the plasmid used in the Special Exercise contained added DNA.

Biology 05LA Fall Quarter 2013

Lab 7 Page 9

Biol 5LA In-class worksheet for Lab 7

Name____________________________

1. Using the worksheet on page 7 of Lab 7: (6 points) a. Calculate the number of transformed cells.

b. Calculate the number of cells available for transformation in each sample (3 drops of competent E. coli cells).

c. Calculate the transformation efficiency.

2. When E. coli cells containing the pUC 118 plasmid are placed on a growth medium containing 10 times as much ampicillin as we used in our laboratory, they are unable to grow. Explain this as best as you can. (1 point)

3. What is the purpose of the (-) control? (1 point)

4. List some advantages for using E. coli as an experimental organism. (1 point)

5. Colonies of transformed E. coli cells (those which are resistant to ampicillin) often are surrounded by satellite colonies of untransformed cells. What is your interpretation of this phenomenon? (Hint: ampicillin does not necessarily kill bacteria; it usually just prevents them from growing. (1 point)