Lab Manual
Introductory Biology (Version 1.4)

© 2013 eScience Labs, LLC All rights reserved www.esciencelabs.com • 888.375.5487

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Table of Contents:
Introduc on:
Lab 1: Lab 2: Lab 3: Lab 4: The Scien fic Method Wri ng a Lab Report Data Measurement Introduc on to the Microscope

Biological Processes:
Lab 5: Lab 6: Lab 7: Lab 8: Lab 9: The Chemistry of Life Diffusion Osmosis Respira on Enzymes

The Cell:
Lab 10: Lab 11: Lab 12: Lab 13: Lab 14: Lab 15: Cell Structure & Func on Mitosis Meiosis DNA & RNA Mendelian Gene cs Popula on Gene cs

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Lab Safety
Always follow the instruc ons in your laboratory manual and these general rules:

eScience Labs, LLC. designs every kit with safety as our top priority. Nonetheless, these are science kits and contain items which must be handled with care. Safety in the laboratory always comes first!

Lab Prepara on
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Please thoroughly read the lab exercise before star ng! If you have any doubt as to what you are supposed to be doing and how to do it safely, please STOP and then:
Double-check the manual instruc ons. Check www.esciencelabs.com for updates and ps. Contact us for technical support by phone at 1-888-ESL-Kits (1-888-375-5487) or by email at Help@esciencelabs.com.

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Read and understand all labels on chemicals.
If you have any ques ons or concerns, refer to the Material Safely Data Sheets (MSDS) available at www.esciencelabs.com. The MSDS lists the dangers, storage requirements, exposure treatment and disposal instruc ons for each chemical.

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Consult your physician if you are pregnant, allergic to chemicals, or have other medical condi ons that may require addi onal protec ve measures.

Proper Lab A re
• •

Remove all loose clothing (jackets, sweatshirts, etc.) and always wear closed-toe shoes. Long hair should be pulled back and secured and all jewelry (rings, watches, necklaces, earrings, bracelets, etc.), should be removed. Safety glasses or goggles should be worn at all mes. In addi on, wearing so contact lenses while conduc ng experiments is discouraged, as they can absorb poten ally harmful chemicals. When handling chemicals, always wear the protec ve goggles, gloves, and apron provided. 5

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Performing the Experiment
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Do not eat, drink, chew gum, apply cosme cs or smoke while conduc ng an experiment. Work in a well ven lated area and monitor experiments at all mes, unless instructed otherwise. When working with chemicals:
Never return unused chemicals to their original container or place chemicals in an unmarked container. Always put lids back onto chemicals immediately a er use. Never ingest chemicals. If this occurs, seek immediate help. Call 911 or “Poison Control” 1-800-222-1222

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Never pipe e anything by mouth. Never leave a heat source una ended.
If there is a fire, evacuate the room immediately and dial 911.

Lab Clean-up and Disposal
• •

If a spill occurs, consult the MSDS to determine how to clean it up. Never pick up broken glassware with your hands. Use a broom and a dustpan and discard in a safe area. Do not use any part of the lab kit as a container for food. Safely dispose of chemicals. If there are any special requirements for disposal, it will be noted in the lab manual. When finished, wash hands and lab equipment thoroughly with soap and water.

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Above all, USE COMMON SENSE!

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Approximate Time and Addi onal Materials Needed for Each Lab
** Note: If you are allergic to nitrile, please contact us and we will send you an alterna ve**

Introduc on:

Lab 1: The Scien fic Method Time: 1 hour Materials: None

Lab 2: Wri ng a Lab Report Time: 1 hour (plus 24 hours prepara on me and 7-10 days for observa on) Materials: Paper towels, water, masking tape Lab 3: Data Measurement Time: 1 hour Materials: Water Lab 4: Introduc on to the Microscope Time: 1 hour Materials: Access to ESL’s Student Portal

Biological Processes:

Lab 5: The Chemistry of Life Time: 1 hour (plus 24 hours prepara on me) Materials: Variety of household substances, plas c wrap, water, cu ng utensil

Lab 6: Diffusion Time: 1.5 hours Materials: Water, watch or mer , viscous liquid from cupboard Lab 7: Osmosis Time: 1 hour (plus 3 hours for observa on) Materials: Water, watch or mer, several types of potatoes, cu ng utensil, paper towel Lab 8: Respira on Time: 1 hour (plus 2 hours prepara on me) Materials: Water, watch or mer, paper towel Lab 9: Enzymes Time: 1 hour (plus 2 hours prepara on me) Materials: Water, watch or mer, string, ice, hot water, paper towel, ginger root, at least 2 other food sources (potato, apple, etc.)

The Cell:

Lab 10: Cell Structure & Func on Time: 1 hour (plus 24 hours for observa on) Materials: Water, square plas c food storage container, mixing bowl, house hold items for use as cell structures (plums, raisins, etc.)

Lab 11: Mitosis Time: 1 hour Materials: None

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Lab 12: Meiosis Time: 1.5 hours Materials: Blue and red markers Lab 13: DNA & RNA Time: 2 hours Materials: Fruit, scissors Lab 14: Mendelian Gene cs Time: 1.5 hours Materials: None Lab 15: Popula on Gene cs Time: 1.5 hours Materials: None

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Addi onal Online Content Found at www.esciencelabs.com

Introduc on:
ESL Safety Video ESL Scien fic Processes Video How Big Is It? Introduc on to the Microscope Measuring Volume Using a Graduated Cylinder Unit Conversions

Log on to the Student Portal using these easy steps: Visit our website, www.esciencelabs.com, and click on the green bu on (says “Register or Login”) on the top right side of the page. From here, you will be taken to a login page. If you are registering your kit code for the first me, click the “create and account” hyperlink. Locate the kit code, located on a label on the inside of the kit box lid. Enter this, along with other requested informa on into the online form to create your user account. Be sure to keep track of your username and password as this is how you will enter the Student Portal for future visits. This establishes your account with the eScience Labs’ Student Portal. Have fun!

Biological Processes:
ESL Biological Processes Video The Structure of an Atom Acid/Base Reac ons Diffusion and Osmosis Tutorial Docking Tutorial

The Cell:
ESL Cell Video Cell Structure Crossword Puzzle Interac ve Videos of Meiosis Interac ve Videos of Mitosis Nature’s Review of RNA DNA Transcrip on & Transla on

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The Cell (con nued):
How Muta ons Work Riken Center’s Developmental Biology Stem Cell Videos A Typical Animal Cell Construc on of the Cell Membrane The Cell Cycle Cell Division DNA Extrac on Virtual Lab

Addi onal Resources:
Stop Watch Conversion Tables

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Lab 1 : Scien fic Method

Concepts to explore:
• • • • • • Testable observa ons Hypothesis Null hypothesis Experimental approach Variables Controls

Concepts to explore:
• • Data collec on Analysis

Introduc on
What is science? You have likely taken several classes throughout your career as a student, and know that it is more than just chapters in a book. Science is a process that uses evidence to understand the history of the natural world and how it works. It is constantly changing as we understand more about the natural world, and con nues to advance the understanding of the universe. Science begins with observa ons that can be measured in some way so that data can be collected in a useful manner by following the scien fic method. Have you ever wondered why the sky is blue or why a plant grows toward a window? If so, you have already taken the first step down the road of discovery. No ma er what the ques on, the scien fic method can help find an answer (or more than one answer!). Following the scien fic method helps to insure scien sts can minimize bias when tes ng a theory. It will help you to collect and organize informa on in a useful way, looking for connec ons and pa erns in the data. As an experimenter, you should use the scien fic method as you conduct the experiments throughout this manual.

Figure 1: The process of the scien fic method

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Lab 1 : Scien fic Method
The scien fic method process begins with the formula on of a hypothesis – a statement of what the experimenter thinks will happen in certain situa ons. A hypothesis is an educated guess – a proposed explana on for an event based on observa on(s). A null hypothesis is a testable statement, that if proven true means the hypothesis was incorrect. Both statements must be testable, but only one can be true. Hypotheses are typically wri en in an if/ then format, such as: Hypothesis: If nutrients are added to soil, then plants grown in it will Figure 2: What affects plant growth? grow faster than plants without added nutrients in the soil. Null hypothesis: If nutrients are added to the soil, then the plants will grow the same as plants in soil without added nutrients. There are o en many ways to test a hypothesis. When designing an experiment to test a hypothesis there are three rules to follow: 1. The experiment must be replicable. 2. Only test one variable at a me. 3. Always include a control.

If plants grow quicker when nutrients are added, then the hypothesis is accepted and the null hypothesis is rejected.

Variables are defined and measurable components of an experiment. Controlling the variables in an experiment allows the scien st to quan tate the changes that occur so that results can be measured and conclusions drawn. There are three types of variables: Independent Variable: The variable that the scien st changes to a predetermined value in order to test the hypothesis. There can only be one independent variable in each experiment in order to pinpoint the change that affects the outcome of the experiment. Dependent Variable: This variable is measured in regards to condi ons of the independent variable—it depends on the independent variable. There can be more than one dependent variable in each experiment.

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Lab 1 : Scien fic Method
Controlled Variable: This variable, or variables (there could be many) reflect the factors that could influence the results of the experiment, but are not the planned changes the scien st is expec ng (by changing the independent variable). These variables must be controlled so that the results can be associated with some change in the independent variable. When designing the experiment, establish a clear and concise procedure. Controls must be iden fied to eliminate compounding changes that can influence the results. O en mes, the hardest part of designing an experiment is not figuring out how to test the one factor you focus on, but in trying to eliminate the o en hidden influences that can skew results. Taking notes when conduc ng an experiment is important, whether it is recording the temperature, humidity, me of day, or another environmental condi on that may have an impact on the results. Also remember that replica on is fundamental to scienfic experiments. Before drawing conclusions, make sure your data is repeatable. In other words, make sure the experiment provides significant results over mul ple trials. O en, the best way to organize data for analysis is as a table or a graph. Remember, any table or graph should be able to stand on its own. In other words, another scien st should be able to pick up the table or graph and have all of the informa on necessary to interpret it, with no other informa on. Table: A well-organized summary of data collected. Only include informa on relevant to the hypothesis (e.g. don’t include the color of the plant because it’s not relevant to what is being tested). Always include a clearly stated tle, label your columns and rows and include the units of measurement. For our example:
Table 1: Plant Growth With and Without Added Nutrients

Variable Control
(without nutrients)

Height Wk1 (mm) 3.4 3.5

Height Wk. 2 (mm) Height Wk. 3 (mm) Height Wk. 4 (mm) 3.6 3.7 3.7 4.1 4.0 4.6

Independent
(with nutrients)

Graph: A visual representa on of the rela onship between the independent and dependent variable. Graphs are useful in iden fying trends and illustra ng findings. Rules to remember:
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The independent variable is always graphed on the x-axis (horizontal), with the dependent variable on the y axis (ver cal). Use appropriate numerical spacing when plo ng the graph, with the lower numbers star ng on both the lower and le hand corners. Always use uniform or logarithmic intervals. For example, if you begin by numbering, 0, 10, 20, do not jump to 25 then to 32.

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•

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Lab 1 : Scien fic Method
•

Title the graph and both the x and y axes such that they correspond to the table from which they come. For example, if you tled your table “Heart rate of those who eat vegetables and those who do not eat vegetables”, be sure to tle the graph the same. Determine the most appropriate type of graph. Typically, line and bar graphs are the most common.

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Line graph: Shows the rela onship between variables using plo ed points that are connected with a line. There must be a direct rela onship and dependence between each point connected. More than one set of data can be presented on a line graph. Figure 3 uses the data from our previous table:

Height (mm)

Figure 3: Plant growth, with and without nutrients, over me

Bar graph: Used to compare results that are independent from each other, as opposed to a con nuous series. Since the results from our previous example are con nuous, they are not appropriate for a bar graph. Figure 4 shows the top speeds of four cars. Since there is no rela onship between each car, each result is independent and a bar graph is appropriate.

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Lab 1 : Scien fic Method

Speed (kph)

Figure 4: Top speed for Cars A, B, C, and D

Interpreta on: Based on the data you collected, is your hypothesis supported or refuted? Based on the data, is the null hypothesis supported or refuted? If the hypothesis is supported, are there other variables which should be examined? For instance, was the amount of water and sunlight consistent between groups of plants - or, were all four cars driven on the same road?

Exercise 1:
Dissolved oxygen is oxygen that is trapped in a fluid, such as water. Since virtually every living organism requires oxygen to survive, it is a necessary component of water systems such as streams, lakes and rivers in order to support aqua c life. The dissolved oxygen is measure in units of ppm—or parts per million. Examine the data in Table 2 showing the amount of dissolved oxygen present and the number of fish observed in the body of water the sample was taken from; finally, answer the ques ons below.

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Lab 1 : Scien fic Method
Table 2: Water Quality vs. Fish Popula on

Dissolved Oxygen (ppm) Number of Fish Observed

0 0

2 1

4 3

6

8

10 13

12 15

14 10

16 12

18 13

10 12

1. Based on the informa on in Table 2, what pa erns do you observe?

2. Develop a hypothesis rela ng to the amount of dissolved oxygen measured in the water sample and the number of fish observed in the body of water.

3. What would your experimental approach be to test this hypothesis?

4. What are the independent and dependent variables?

5. What would be your control?

6. What type of graph would be appropriate for this data set? Why?

7. Graph the data from the table above.

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Lab 1 : Scien fic Method
8. Interpret the data from the graph made in Ques on 7.

Exercise 2:
Determine which of the following observa ons are testable. For those that are testable: • Write a hypothesis and null hypothesis • What would be your experimental approach? • What are the dependent and independent variables? • What is your control? • How will you collect your data? • How will you present your data (charts, graphs, types)? • How will you analyze your data?

1. When a plant is placed on a window sill, it grows faster than when it is placed on a coffee table in the middle of the living room.

2. The teller at the bank with brown hair and brown eyes and is taller than the other tellers.

3. I caught four fish at the seven o’clock in the morning but didn’t catch any at noon.

4. The salaries at Smith and Company are based on the number of sales and Billy makes 3,000 dollars more than Joe.

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Lab 1 : Scien fic Method
5. When Sally eats healthy foods and exercises regularly, her blood pressure is lower than when she does not exercise and eats fa y foods.

6. The Italian restaurant across the street closes at 9 pm but the one two blocks away closes at 10 pm.

7. Bob bought a new blue shirt with a golf club on the back for twenty dollars.

8. For the past two days the clouds have come out at 3 pm and it has started raining at 3:15 pm.

9. George did not sleep at all last night because he was up finishing his paper.

10. Ice cream melts faster on a warm summer day than on a cold winter day.

11. How can you apply scien fic method to an everyday problem? Give one example.

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Introduc on

Lab 2 Wri ng a Lab Report
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Lab 2: Wri ng a Lab Report

Concepts to explore:
• • • What is a lab report? The parts of a lab report How to write a lab report

Introduc on
A lab report is a scien fic paper describing an experiment, how it was done and the results of the study. Experiments are performed to test whether what one thinks may happen, actually does. The lab report lays out the results of the experiment and can be used to communicate the findings to other sciensts. It allows the findings of one scien st to be examined, replicated, refuted or supported by another scien st. Though most lab reports go unpublished, it is important to write a report that accurately characterizes the experiment performed. Even if what is described never reaches the public or the scien fic community, the report lays the founda on for other experiments. It also provides a wri en record of what was done, so that others can understand what the inves gator was thinking and doing.

Figure 1: Lab reports are an essen al part of science, providing a means of repor ng experimental find-

Parts of a Lab Report: Title: A short statement summarizing the topic of the report. Abstract: A brief summary of the methods, results and conclusions. It should not exceed 200 words and should be the last part wri en. Introduc on: This is an overview of why the experiment was conducted. There are three key parts: Background: Provides an overview of what is already known and what ques ons remain unresolved regarding the topic of the experiment. Assume the reader needs a basic introduc on to

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Lab 2: Wri ng a Lab Report the topic and provide the informa on necessary for them to understand why and how the experiment was performed. Objec ve: Explain the purpose of the experiment. For example; “I want to determine if taking baby aspirin every day prevents second heart a acks”. Hypothesis: This is your “guess” as to what will happen when you do the experiment.

Materials and Methods: These are detailed descrip ons of what was used Figure 2: Follow the guideto conduct the experiment, what was actually done (step by step) and how lines in this introduc on it was done. The descrip on should be exact enough that someone readwhen wri ng a lab report. ing the report can replicate the experiment. Make sure to include all the equipment and supplies used, even they seem obvious and did not seem to play a large role. When describing the methods, go in order from the first step to the last. Do not list the procedures used in a numerical fashion, but write them in complete sentences and paragraphs, much like you would if speaking. Results: This is the data obtained from the experiment. This sec on should be clear, concise and to the point. In this sec on tables and graphs are o en appropriate and frequently are the best way to present the data. Do not include any interpreta ons, only the raw data. Discussion: This is where the scien st (you) can interpret the data you obtained and draw conclusions. Was your hypothesis (“guess”) supported or refuted? Discuss what these findings mean, look at common themes, rela onships and points that perhaps generate more ques ons. If fewer second heart a acks were reported when baby aspirin was taken, but only in women, this would lead to addi onal ques ons. When appropriate, discuss outside factors (i.e. temperature, me of day, etc.) that may have played a role in the experiment and what could be done to control those in future experiments. Conclusion: A short, pointed summary that states what has been learned from this experiment. References: Any ar cles, books, magazines, interviews, newspapers, etc., that were used to support your experimental protocols, discussions and conclusions, should be cited in this sec on.

Important Points to Keep in Mind • •
Do not confuse the sec ons of your paper. Pay a en on to the difference between the results and discussion sec on. Be clear, concise and complete.

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Lab 2: Wri ng a Lab Report
• • •
If your results are inconclusive, as are most experiments, say so. Proof read your report. A lab report is expected to be able to withstand scru ny. Do not plagiarize; give credit to all references used.

Experiment 1: Design an Experiment
The following experiment is meant to be designed by you! With the beans provided in the kit, you will design and execute an experiment to test several factors that influence seed germina on. Whatever your experimental design, be sure to include controls and make sure it is reproducible!

Materials
100 beans 10 5 x 8in bags Permanent marker Ruler Paper towels* Water* Masking tape* *You must provide

Notes about bean germina on:

• • •

The me to germina on will decrease if you soak the beans overnight It may take 7-10 days for the beans to ‘sprout’ Make sure the paper towels remain moist for the dura on of your experiment

Proce1. Think of 10-20 variables that may affect seed germina on, recording them in Table 3.

dure

2. From your list of variables in Table 3, select three to test. Form a hypothesis for why each affects seed germina on. 3. To germinate the beans, place one folded paper towel, moistened but not soaking wet, into the 5 x 8in bag. Place 10 beans in a horizontal line on the paper towel (between the paper towel and bag). 4. Label each bag with the variable being tested.

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Lab 2: Wri ng a Lab Report
5. Hang each bag ver cally using masking tape in the environment you select. 6. Create a table for your data, including tle, units, and any other useful informa on. 7. Select the appropriate type of graph, and report the data you collected. 8. Write a lab report for this experiment in the space provided.
Table 3: Variables That May Influence Seed Germina on

Variable

Hypothesized Effect

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Lab 2: Wri ng a Lab Report

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Lab 2: Wri ng a Lab Report

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Introduc on

Lab 3 Data Measurement
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Lab 3: Data Measurement

Concepts to explore:
• • • The metric system Conver ng units Techniques for obtaining accurate measurements

Introduc on
Biology relies heavily on the use of numbers, measurements and calcula ons. Consequently, scien sts use a universal measuring standard called the metric system. Because the metric system is based on units of ten, it simplifies making conversions within that system. The basic units of measurement in the metric system are:
Note: In the table below meters are shown as an example. The prefixes remain the same with liter or gram.

Meter: when measuring distance. Each basic unit can be divided or expanded upon using the following prefixes: Prefix Nano (n) Micro (µ) Milli (m) Cen (c) Deci (d) Prefix Deka (da) Hecto (h) Kilo (k) Mega (M) Giga (G) Abbrevia on 10-9 10 10
-6 -3 -2 -1

• • •

Gram: when measuring mass. Liter: when measuring liquid volume.

Mul plier used to convert TO meters 0.000000001 0.000001 0.001 0.01 0.1 Mul plier used to convert TO meters 10 100 1000 1000000 1000000000

Mul plier used to convert FROM meters 1000000000 1000000 1000 100 10 Mul plier used to convert FROM meters 0.1 0.01 0.001 0.000001 0.000000001

10 10

Abbrevia on 101 102 10 10 10
3 6 9

To convert between units, mul ply using the conversions above (conversions can also be made by division, though not with this table). 31

Lab 3: Data Measurement

Mul plica on Example:

•

To convert 200 meters (m) to kilometers (km):

mul ply 200 m x 0.001 = .2 km

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To convert 450 millimeters (mm) to meters (m): mul ply 450 mm x 0 .001 = .45 m

Figure 1: accurate data measurement is key to reproducible science.

When conver ng from units less than a meter to greater than a meter (or the other way around), first convert to a meter and then to the final unit. To convert 40,000 cm to kilometers:

• • •

mul ply 40,000 cm x 0.01 = 400 m mul ply 400 m x 0.001 = 0.4 km 40,000 cm = 0.4km

Exercise: 1) Convert the following: 3 m = __________ cm 83 m = __________ µm 41,692 m = __________ mm 110 kilometers = __________ m = ____________ mm 3.7 hectometers =_________ m =____________ cm 451,000,000 µm = _________ m = ___________ dam

2) Imagine a field is about 100 meters long. If you run a 5K race how many meters is it? Approximately how many “fields” does this equate to?

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Lab 3: Data Measurement
Length, Area, Volume, Mass and Temperature Length is measured in meters. The area of a square or rectangle is measured by mul plying length (in meters) by width (in meters). The unit of measurement is m2, which reads “meters squared” or “square meters”. When you see this nota on, it is an indica on that the measurement is describing area. Example: If a box is 12 cm long and 24 cm wide, its area is: 12 cm X 24 cm = 288 cm2 Volume can be measured by mul plying length (m) by width (m) by height (m). The unit of measurement is m3, which reads “meters cubed” or “cubic meters”. When you see this nota on, it is an indicaon that the measurement is describing volume. Example: If the same box is 4 cm high, its volume would be: 12 cm X 24 cm X 4 cm = 1,152 cm3 To convert this volume this to meters, 1,152 cm3 must be mul plied by 0.001. 0.001 is used because the number reflects a volume measurement (cubic meters) rather than an area measurement (square meters): 1,152 cm3 X 0.001 = 1.152 x 10-3 m3

Exercise: 3) Measure the following objects.

A) Your computer screen (in meters) Length______________ Width ______________ Area _______________ Volume ____________ B) A 100 mL beaker: (in millimeters) Length _____________ Width _____________ Area ______________ Volume ___________

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Lab 3: Data Measurement
C) Your lab kit box lid: (in cen meters) Length _____________ Width _____________ Area _______________ Volume ____________
Figure 2: Be aware of the margin of error possible with instruments

Mass is the amount of ma er an object possesses. It is the metric systems measurement of weight and is expressed in grams (g). When using instruments, such as a scale, there is always a margin of error. This is a result of either human or mechanical error. Therefore, it is prudent to perform measurements at least three mes to find the average (most precise) measurement. Exercise: 4) Determine the mass of the objects listed below (in grams). Pay a en on to the units. Since you do not have a metric scale, we will provide you data to work with. A) Baseball Mass (measurement 1): ____.145__kg Mass (measurement 2): ____145.05_ g Mass (measurement 3): 145,750.77 mg Mass (average): __________g Convert: ___________kg B) Piece of fruit Mass (measurement 1): ____310____ g Mass (measurement 2): ___0.318____kg Mass (measurement 3): __309,143___ mg Mass (average): __________cg Convert: ___________ g

Volume is a three dimensional measurement of how space is occupied. Previously we expressed vol-

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Lab 3: Data Measurement ume in m3 (cubic meters). However, the measurement can be reported in units of cubic length or liters. To convert from one to the other, the conversion 1 cm3 = 1ml is used. Example: To determine the volume of a measurable object, mul ply length x width x height. If a wooden block is 15 cm long, 20 cm wide and 4 cm high, the volume can be found by: Volume = 15 cm x 20 cm x 4 cm = 1,200 cm3 = 1,200 ml = 1.2 L

NOTE: When an object is solid and does not have measurable sides (i.e. a solid marble), water displacement can be used to determine the volume.

A graduated cylinder is o en used to measure volumes. The graduated cylinder is filled with water and this ini al volume is recorded. The object is added carefully and the new volume is recorded. The difference of these two volumes is the volume of the object! Ex: The ini al water level in a graduated cylinder is 25.8mL. A er an irregularly shaped object is placed into the cylinder, the water level reads 42.9mL. What is the volume of the irregularly shaped object? Answer: 17.1mL or 17.1cm3

When measuring a liquid there is a certain place that one must measure - the bo om of the meniscus. The meniscus is the curved line that a liquid makes when placed in a narrow container. When looking for the bo om of the meniscus, one must look straight at it. When one’s line of sight is too high, then the reading that is received is too low. When one’s line of sight is too low, then the reading received is too high.

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Lab 3: Data Measurement
Exercise: Determine the volume of the following objects. If you cannot do so by measuring the dimensions, use a different technique. A) The chemical box inside of your kit: Length: ________ m ________ cm Width: _________ m _________ cm Height: _________ m _________ cm Volume: ________ L

B) Test tube: Length: _________m ________ cm Width: __________ m ________ cm Height: __________ m ________ cm Volume: _________ L

C) Pick an object from your home. Object:______________. Length: _________ m Width: __________ m Height: __________ m Volume: _________ L

Exercise 1. If you want to determine the volume of a swimming pool, name two ways you could do this.

2. Measure the volume of a soup bowl from your cupboard. Volume: _________ mL

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Lab 3: Data Measurement
Temperature is a measure of the amount of heat present in an object. We use the Fahrenheit scale in the U.S., but the scien fic standard is Celsius. In Celsius, water boils at 100o C and freezes at 0o C. To convert between Fahrenheit and Celsius, use the following equa on: o C = 5/9 (o F – 32o)

Example: the human body has a temperature of 98.6o F: o C = 5/9 (98.6o F – 32o) C = 37

o

Exercise: 1. Convert the following: 121 o F = _______________ o C 32o F = ________________ o C 0 o F = ________________ o C 77 o F = ________________ o C

2. With your thermometer, measure the temperature of the following objects:

A) Glass of cold tap water: _______________ o C B) Your kitchen: _______________ o C C) Inside your freezer: _______________ o C D) Palm of your hand (wrap your hand around the thermometer, but do not squeeze): _____________ o C

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Introduc on

Lab 4 Introduc on to the Microscope
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Lab 4: Introduc on to the Microscope

Concepts to explore:
• • • • • Types of microscopes Parts of a microscope How to use a microscope Preparing a wet mount slide Depth of field

Introduc on
Some objects are far too small to be seen with the human eye. However, by using a microscope many can be viewed in great detail. There are many types of microscopes that range from low–level magnifica on (i.e., hand-held magnifica on lens) to very high-power magnifica on (i.e., an electron microscope). In the middle of that range lies the light microscope, or for our purposes, the compound light microscope, which uses mul ple lenses. The compound light microscope (Figure 1) has two sets of lenses:

• •

the ocular lenses (close to your eyes) the objec ve lenses (close to the “object” on the stage).

Along with a light source, these lenses work together to magnify the object being viewed. In the case of the compound light microscope, the total magnifica on is equal to the magnifica on power of the ocular lens mul plied by the magnifica on power of the objec ve lens. For example, if the ocular lens magnifies 10X (this means 10 mes) and the objec ve lens magnifies 10X, the total magnifica on is 100X.

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Lab 4: Introduc on to the Microscope

Figure 1: A compound microscope can magnify objects that are not visible to the naked eye so that they can be studied.

Parts of a Compound Light Microscope Base: The flat support of the microscope. Light: Illuminates the object being viewed. This can be either in the form of a light source or a mirror that reflects ambient light onto the image. In the la er case it is important to be working in an environment with adequate ambient light. Stage: Supports the slide or other material to be viewed. Diaphragm: Controls the amount of light allowed on the object. Stage Clips: Secure the slide in place. Revolving Nosepiece: Rotates the objec ve lenses of different magnifica ons and allows one of them to be posi oned over the slide.

42

Lab 4: Introduc on to the Microscope
Arm: Connects the lower base and the upper head of the microscope (also used to carry the microscope). Head: Supports both the ocular lens and the revolving nosepiece. Ocular Lens (eyepiece): The lenses on the microscope typically have a magnifica on of 10X. If your microscope has a pointer, which is used to indicate a specific area of the specimen, it is a ached here. Types of Microscopes Monocular Microscope: Has a single ocular eyepiece. Binocular Microscope: Has two ocular eyepieces. How to Use a Microscope 1. Always carry a microscope with one hand securely around the arm and the other underneath the base for support. 2. Place the microscope on a table, plug it in, and turn on the light source (or adjust the mirror as necessary). Note: When cleaning a microscope, do not use paper towels or cloths as this will scratch the lens. To preserve the microscope, use only lens paper that will not scratch the op cs. 3. To prevent damage to the lens or slides, always start and end with the scanning power objec ve lens (the shortest one) above the light source. 4. Place your slide on the stage and secure it with the stage clips. It is helpful to visually orient the slide so the object to be viewed is directly in the middle of the opening in the stage where the light is directed up toward the slide. 5. Turn the course adjustment knob to bring the stage all the way up to the scanning power objec ve lens. While looking through the lens, use the course adjustment knob to slowly lower the stage un l the specimen comes into focus. Note: When using a binocular microscope, adjust the distance between the two oculars un l only one object is seen. Record this distance and set your microscope to this distance every me you use it. If someone else uses the microscope, the lenses may be re-adjusted for their eyes. 6. To adjust the light, open or close the diaphragm located over the light source. When properly illuminated, the specimen should not be gray or excep onally bright.

43

Lab 4: Introduc on to the Microscope
7. With the object is in general focus, rotate the revolving nosepiece to the low-power lens (the next longest). A er focusing with the course adjustment knob, switch to the fine adjustment knob to obtain more precise and greater detail. It may also be necessary to adjust the light, because more light reduces contrast (sharpness).

8. To become familiar with the mechanical stage knobs around the base of the microscope (if present), turn one slowly to the right, no ng that the image will be moving toward the le . This image inversion is caused by the lenses. 9. If you need higher magnifica on, slowly rotate the high-power lens into place (the next longest lens). This will bring the p of the lenses very close to the slide. 10. Make sure the objec ve lens does not touch the slide. 11. Whenever you use the high-power lens, only use the fine adjustment knob. If the object was well focused while viewing with the low-power lens, very li le adjustment should be necessary. 12. If you cannot bring the object into focus, return to the low-power lens, focus the object, and then return to the high-power lens. 13. When finished, move the revolving nosepiece to the scanning objec ve lens posi on before removing the slide.

How to Prepare a Wet Mount Slide 1. To make a wet mount for a specimen that is not already in liquid, take a clean slide and place the specimen in the center. 2. Add a drop of water. Note: For cells that are transparent, it may be necessary to add a small drop of stain as opposed to water. 3. Carefully add a coverslip by placing one end down and slowly lowering the other end. Note: If the coverslip is added too quickly, large air bubbles may become trapped which can cause difficulty viewing the slide. If this happens, gently remove the coverslip, add another drop of water and try again. 4. Remove excess liquid on the bo om of the slide or around the edges before it is placed on the microscope to avoid damage to the lens. Just touch a ssue to the edge of the

44

Lab 4: Introduc on to the Microscope coverslip to draw away the water (this is called diffusion and there will be a lab on diffusion later in the series).

5. If the specimen is already in liquid, place a drop in the middle of the slide and add the coverslip as you would for a dry specimen.

Experiment 1: Virtual Magnifica on Exercise

Materials •
“How Big Is It?” demonstra on on Student Portal

Note: Review the direc ons for signing in to the Student Portal at the beginning of this manual if uncertain how to access this informa on

Procedure
1. Log into your eScience Student Portal account and locate the “How Big Is It” demonstraon under the Introduc on sec on. 2. Load the anima on and beginning with the head of a pin, increase the magnifica on by clicking the arrows below the picture. Note the rela ve sizes of the objects on the pinhead. 3. Be sure to no ce the magnifica on bar on the lower por on of the demonstra on that shows the magnifica on required to see the objects.

Ques ons
1. At what magnifica on do you first no ce the ragweed pollen?

45

Lab 4: Introduc on to the Microscope
2. Which is bigger, a rhinovirus or E. Coli?

3. Based on the magnifica on, how many of the E. Coli can fit into the same space as the head of a pin?

4. About how many red blood cells could fit across the diameter of a human hair (again, look at the magnifica on scale)?

Experiment 2: Virtual Microscope

Materials •Virtual Microscope on Student Portal

Note: Review the direc ons for signing in to the Student Portal at the beginning of this manual if uncertain how to access this informa on

Procedure
1. Log into your eScience Student Portal account and locate the Virtual Microscope ac vity under the Online Learning Tools sec on. Click the link so that the Virtual Microscope opens in a new window.

46

Lab 4: Introduc on to the Microscope
2. Take a tour of the virtual microscope by clicking the “Start Tour” bu on on the right hand side and learn how to use the different controls to effec vely use the simula on. 3. Once you are comfortable using the virtual microscope, switch views so that you are looking through the oculars and can look at the slides (be sure your light is on before you do this or you will have to switch views to hit the power bu on!). 4. Select the le er e slide on the top right of the page and examine with the 4X objec ve. Increase the magnifica on un l the le er no longer fits in the field of view. Note this magnifica on. 5. Select the cheek smear slide on the right side of the page and bring the cells within the red circle into focus using the 4X, then 10X objec ves. 6. View the slide under 40X and 100X objec ves, making sure you stay within the red circle. 7. Next, select the onion root p slide on the right side of the page to view. Start with the 4X objec ve, and bring the cells within the red circle into focus. 8. Switch to the 10X objec ve and readjust the focus so the slide is clear. Con nue looking at the slide under higher magnifica on using the 40X and 100X objec ves. 9. On the le hand side of the screen, select the “Try This” box. Under measurement, select the m1 box to open an ac vity that will instruct you how to measure the le er e. (Remember this number as you will have to report it in Ques on 5!) Note—do not click on the “Try This” instruc on box or the so ware may freeze. If this happens, refresh your browser and start again. You will have to go through the tutorial tour again in order for the “Try This” box to become available.

Ques ons
1. What is the first step normally taken when you look through the oculars?

2. What is the highest objec ve you can use to see the en re le er e?

47

Lab 4: Introduc on to the Microscope

3. The nuclei of the cheek cells have been stained using a special dye so that they appear purple. What shape are they?

4. At high magnifica on, you may no ce that not all of the nuclei in the onion root p slide appear as the shape you described in the ques on above. What do they look like?

5. A er comple ng the m1 exercise in the “Try this” sec on, how tall is the le er e?

48

Biological Processes

Lab 5 Chemistry of Life

Lab 5: Chemistry of Life

Concepts to explore:
• • • • • • Atoms Elements Compounds Chemical bonds Molecules/Macromolecules Energy and metabolism

Concepts to explore:
• • Acids and bases The effects of surface area and volume

Introduc on
It is important to have a general understanding of chemistry before you can begin to understand how living organisms manage to reproduce, grow, move, eat, and perform a great many more func ons. To begin understanding the myriad of reacons that occur within a cell, it is important to review the basics of chemistry. Recall that anything that occupies space and has mass is called ma er; all ma er is made of atoms. Atoms are made of a nucleus and two kinds of subatomic par cles: electrons (nega vely charged par cles), and protons (posi vely charged par cles). Elements are pure substances that are made of only one type of atom. More than 90% of ma er is composed of combina ons of just four elements: oxygen, carbon, hydrogen, and nitrogen. There are over 100 elements known, each with different properRemember: Mass is the quan ty of ma er an object has; weight is the force produced by gravity ac ng on the mass of an object

Figure 1: The periodic table of elements categorizes all of the known elements

51

Lab 5: Chemistry of Life es. The periodic table has been used to categorize these elements. In nature, most elements are not found alone; atoms of most elements combine with the same or different elements to make compounds. A compound is a mixture of two or more elements in definite propor ons. These atoms are held together by chemical bonds, bringing them to a stable state. Chemical bonds also store energy. The two most common bonds are covalent bonds and ionic bonds. Covalent bonds form when two atoms share electrons. The simplest part of a substance that retains the proper es of that substance is called a molecule. Ionic bonds form when an atom or molecule carries an electrical charge, which a racts an atom or molecule of the opposite charge. Very large molecules are termed macromolecules. All living organisms use the same four types of macromolecules for cellular metab- Figure 2: Have you ever drank orolisms and reproduc on. These common biological macromolecules ange juice right a er brushing your are proteins, nucleic acids, carbohydrates, and lipids. The proper es teeth? Yuck! The displeasing taste is a result of the acid/base reac on they convey are of great importance to cell func on, and you will that occurs when a weak acid learn about each in future labs.
(orange juice) mixes with a weak

Chemical reac ons take one or more substance and change it to base (toothpaste). create a new substance. This requires energy. When chemical bonds are broken, energy is made available for the reac on to proceed. Most reac ons also require energy to ini ate the reac on. This is called the ac va on energy, and it differs for each reac on. Catalysts are chemicals that lower the ac va on energy. You will learn about biological catalysts called enzymes later in this manual. Living things require a constant supply of energy. Throughout this manual, you will learn about the reac ons that take place inside of organisms. The sum of these reac ons is called metabolism, and is a general term used to describe the energy require to keep those reac ons occurring. Two important classes of compounds are acids and bases. Both have physical and chemical differences that can be observed and tested. Acids ionize in water to produce a hydronium ion (H3O+) and bases dissociate in water to produce a hydroxide ion (OH-). A compound’s acidity or alkalinity (how basic it is) can be measured on a scale called pH. The pH of a substance is a measure of the concentra on of hydronium ions. A solu on that contains a lot of hydronium ions but few hydroxide ions is considered to be very acidic. In contrast, a solu on that contains many hydroxide ions but few hydronium ions is considered to be very basic. pH values range from 1-14, with 1 being highly acidic, 14 highly basic, and 7 neutral. Have you ever wondered why cells are the size they are? There are many reasons, but one important one is the surface area to volume ra o. In subsequent labs, you will learn how cells divide once they

52

Lab 5: Chemistry of Life reach a cri cal size. Nutrients and oxygen need to diffuse through the cell, and waste needs to diffuse out of the cell. This must happen quickly for the cell to survive – which happens when the surface area to volume ra o of the cell is high.

Experiment 1: What Household Substances are Acidic or Basic?
There are chemicals, called pH indicators, which change color when they come into contact with an acid or a base. In the following experiment, you will be using pH paper to determine the pH of various household substances. The key below indicates the color the paper turns as a func on of the pH. In this way, pH paper allows scien sts to determine to what degree a substance is acidic or basic and can provide an approximate pH value.

Materials
10 pH paper strips 5mL Vinegar (15,000g/mol) Iodine-Potassium iodide (IKI) 4 Glucose test strips

15cm Dialysis tubing** 4 100mL Beakers 4 Small rubber bands 7 Graduated pipe es Water* Watch* *You must provide ** Cut to exact length

• • •

Note: You will need dialysis tubing in subsequent experiments, so be sure to cut the amount specified in the direc ons. Dialysis tubing can be rinsed and used again if you make a mistake. Dialysis tubing must be soaked in water before you will be able to open it up to create the dialysis “bag”. Follow the direc ons for the experiment, beginning with soaking the tubing in a beaker of water. Then, place the dialysis tubing between your thumb and forefinger and rub the two digits together in a shearing manner. This should open up the "tube" so you can fill it with the different solu ons.

Procedure
1. Fill one 100mL beaker with 50mL water and submerge the dialysis tube for 10 minutes. Fill a second beaker with 80mL water (this is the one you will put the filled dialysis bag into in Step 9). 2. A er the ten minutes have passed, remove the dialysis tube and close one end by folding over 3cm of one end (bo om). Fold it again and secure with a rubber band (use two if necessary). 3. Make sure the closed end will not allow a solu on to leak out. You can test this by adding a few drops of water and looking for leakage. Pour the water out before con nuing.

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Lab 6: Diffusion

A en on! Do not allow the open end of the bag to fall into the beaker. If it does, remove the tube and rinse thoroughly with water before refilling with a starch/glucose solu on and replacing it in the beaker.

Figure 2: Experimental set-up

4. Use a graduated pipe e to add 5ml of glucose solu on to a third beaker and label it “Dialysis bag solu on”. Using another graduated pipe e, add 5mL of starch solu on to the same beaker. Mix by pipe ng the solu ons up and down the pipe e six mes. 5. Transfer 8mL of the dialysis bag solu on (glucose and starch) into the prepared dialysis bag. The remaining 2mL will serve as a sample to test for the presence of glucose and starch (to act as a control and show that both glucose and starch were present in the solu on poured into the dialysis bag).

Indicator Reagents
IKI Solu on: Yellow = no starch Purple/Black = starch Glucose Test Strip: Yellow = no glucose

Green = glucose 6. Label the last (fourth) beaker “Beaker solu on”, and using a clean pipe e, transfer a sample of 2mL of the water in the second beaker to this beaker (to act as another control to show that the water the dialysis bag is placed in does not contain starch or glucose). 7. Test for the presence of glucose by dipping one glucose test strip into the dialysis bag solu on sample (third beaker) and another strip into the beaker solu on sample (fourth beaker). Wait 1 minute, then observe the color of the test strip. Record your results in the following tables (Tables 4 and 5). 8. Next, add a few drops of IKI solu on into both sample beakers (the third and fourth beakers). Record your observa ons in Tables 4 and 5. 9. Place the filled dialysis tube into the second beaker filled with 80mL of water with the open end draped over the edge of the beaker as shown below. 10. Use a pipe e to add 2ml of IKI solu on to the beaker water. Record the ini al color of both the beaker water and the solu on in the dialysis tube in the table below (Table 4). 11. A er the solu on has diffused for 60 minutes, remove the dialysis tube from the beaker.

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Lab 6: Diffusion
12. Again, test for the presence of glucose by dipping one glucose test strip into the dialysis bag directly and another strip into the beaker solu on. Again, wait one minute before reading the results of the test strip. Record your results for the presence of glucose and starch in the following Tables 4 and 5.
Table 4: Starch Diffusion

Ini al color Beaker Dialysis tube

Starch present?

Final color

Starch present?

Table 5: Glucose Diffusion

Present/Absent Before Dialysis Beaker Dialysis tube

Present/Absent A er Dialysis

Ques ons
1. Which substance crossed the dialysis membrane? What evidence from your results proves this?

2.

What molecules remained inside of the dialysis bag?

3.

Of the substances that diffused through the bag, did all of the molecules diffuse out?

4.

Does the dialysis bag or the beaker contain more starch? What about glucose?

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Lab 6: Diffusion
5. Is the bag hypotonic with regards to the IKI solu on, or the beaker? What about the starch solu on?

6.

What results would you expect if the experiment started with glucose and IKI Solu on inside of the bag, and starch and water in the beaker? Why?

7.

Draw a diagram of this set up. Use arrows to depict the movement of each substance in the dialysis bag and the beaker.

8.

What type of membrane does the dialysis tubing represent? Give an example of this type of membrane that can be found inside the body.

9.

How does the glucose concentra on affect diffusion rate?

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Biological Processes

Lab 7 Osmosis
71

72

Lab 7: Osmosis

Concepts to explore:
• • • • • Osmosis Hypertonic Hypotonic Isotonic Osmo c pressure

Introduc on
A major determinant of diffusion in a biological system is membrane permeability. Small, uncharged molecules pass through cellular membranes easily, while most and/or charged molecules cannot pass through the membrane. The movement of water across a selec vely permeable membrane, like the plasma membrane of the cell, is called osmosis. Osmosis occurs when a membrane separates solu ons of different concentraons. The membrane allows the solvent to pass through, but not the solutes. Ul mately, membrane selec vity and the movement of water in and out of the cell regulates the concentra on of intracellular material. Remember, a solu on contains two or more substances (solutes) that have been dissolved by a solvent. In the context of a cell, the intracellular and extracellular fluids are the solvents which contain dissolved material (solutes). As solute concentra on increases, solvent concentra on decreases. Tonicity is a rela ve term used to describe osmo c pressure of two solu ons separated by a semipermeable membrane. It is influenced by solutes that cannot cross the semipermeable membrane (solutes that do cross the membrane will always move to achieve equal concentra ons on both sides of the membrane). Thus, tonicity determines the net direc on of movement of water molecules.
Figure 1 The three types of tonicity

73

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Lab 7: Osmosis
There are three types of tonicity (Figure 1) —remember that it is a rela ve term used to compare one solu on to another:

• • •

A hypertonic solu on contains a greater concentra on of solutes unable to cross the membrane compared to the solu on on the other side of the membrane. A hypotonic solu on contains a lower concentra on of solutes unable to cross the membrane compared to the solu on on the other side of the membrane. An isotonic solu on has an equal concentra on of impermeable solutes on both sides of the semi-permeable membrane.

When osmosis takes place, water flows from hypotonic solu ons to hypertonic solu ons, un l the solu ons become isotonic. In most biological systems, cells are hypertonic and extracellular water flows into them. If placed in pure water, they will burst (lyse) as a result of the increased pressure on the membrane from the addi onal water that diffused into the cell. Osmo c pressure (the force required to prevent osmosis) is directly correlated with tonicity (higher tonicity causes an increase in osmo c pressure). Some cells, such as plant cells, have specialized structures that regulate osmo c pressure and prevent lysis.

Experiment 1: Direc on and Concentra on Gradients
In this experiment, we will inves gate the effect of solute concentra on on osmosis. A semi-permeable membrane (dialysis tubing) and sucrose will create an osmo c environment similar to that of a cell. Using different concentra ons of sucrose (which is unable to cross the membrane) will allow us to examine the net movement of water across the membrane.

Materials
30% Sucrose solu on 4 15cm Pieces dialysis tubing** 3 250mL Beakers 8 Rubber bands 10mL Graduated cylinder Blue, red, yellow, green beads

Concepts to explore:
Water* Watch* *You must provide **Cut to exact length

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Lab 7: Osmosis

• • •

Note: You may need dialysis tubing in subsequent experiments, so be sure to cut the amount specified in the direc ons. Dialysis tubing can be rinsed and used again if you make a mistake. Dialysis tubing must be soaked in water before you will be able to open it up to create the dialysis “bag”. Follow the direc ons for the experiment, beginning with soaking the tubing in a beaker of water. Then, place the dialysis tubing between your thumb and forefinger and rub the two digits together in a shearing manner. This should open up the "tube" so you can fill it with the different solu ons.

Procedure
1. Submerge the four pieces of dialysis tubing into a 250 mL beaker filled with 100 ml of water for at least 10 minutes. 2. A er 10 minutes, remove one piece of tubing from the beaker. On one end (not the whole tube), gently twirl the tubing into a long, thin cylindrical piece that is able to fit into the hole of the yellow bead. 3. Insert the long cylindrical end of the tube into the center hole in the yellow bead. Once it is through, pull the cylindrical end un l there is about 1.5 to 2cm of tubing extending beyond the bead

Figure 2: Fold the bag un l you have a piece narrow enough to be threaded through the bead.

4. Take the extra tubing you just pulled through the bead and fold it back over the bead, towards the remaining, non folded tube. Place a rubber band above the bead and around the extra tubing as to be sure no solu on can leak out of the tube (see Figure 2).

Figure 3: Beads help to secure the ends of the dialysis bags and iden fy each one.

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Lab 7: Osmosis
•
To test that no solu on can leak out, add a few drops of water and look for water leakage. Make sure you pour the water out before con nuing to the next step.

5. Repeat steps 2-4 with the three remaining dialysis tubes, using each of the three remaining bead colors (Figure 3).
6. Table 1 provides a dis nc on as to what bead belongs to which tube. Using a 10mL graduated

cylinder, measure and fill the appropriate dialysis bag with the designated concentra on of sucrose solu on (3%, 15% or 30%) by adding the volumes of sucrose and water listed in the table below.
Table 1: How to Make a Serial Dilu on of Sucrose

Bead Color Yellow Red Blue Green 7. 8.

Bag Number Bag #1: 30% sucrose Bag #2: 15% sucrose Bag #3: 3% sucrose Bag #4: 3% sucrose

Stock Sucrose Solu on 10mLs 5mLs 1mL 1mL

Water 0mLs 5mLs 9mLs 9mLs

Rinse the outside of the bags with water to remove any remaining sucrose. Pour 150mL of the stock sucrose solu on (30%) into the 250mL beaker (beaker #1). Using the graduated cylinder, measure 20mLs of the stock sucrose solu on and 180mL of water to create a 3% sucrose solu on and place it into the 250mL beaker (beaker #2). Place bags #1-3 (red, blue, yellow) into beaker 2 and bag #4 (green) into beaker 1 (Figure 4).

9.

Figure 4: The dialysis bags are filled with varying concentraons of sucrose soluon and placed in one of two beakers.

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Lab 7: Osmosis
10. In Table 2, predict whether water will flow in or out of each dialysis bag. 11. Allow the bags to sit for one hour. While wai ng, dump out the water in the 250 mL beaker that was used to soak the dialysis tubing in step 1. We will use this in the last part of the experiment. 12. A er allowing the bags to sit for one hour, remove them from the beakers. 13. Carefully open the bags, no ng that o en mes the tops may need to be cut as they tend to dry out. Measure the solu on volumes of each dialysis bag using the empty 250 ml beaker. Record your data in Table 2.
Table 2: Water Movement

Ini al Volume Bag#1 10mL Bag #2 10mL Bag #3 10mL Bag #4 10mL

Sucrose %

Predic on: Will water move in or out?

Final Volume

Ques ons
1. For each of the bags, iden fy whether the solu on inside was hypertonic, hypotonic or isotonic in comparison to the beaker solu on it was placed in.

2. Which bag increased the most in volume? Why?

3. What does this tell you about the rela ve tonicity between the contents of the bag and the solu on in the beaker?

4. What would happen if bag 1 is placed in a beaker of dis lled water?

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Lab 7: Osmosis
5. Osmosis is one way that your body balances water concentrations within and outside your bloodstream. Explain how you think osmosis works in terms of tonicity in the body.

Experiment 2: Tonicity and the plant cell
Plant cells are able to generate osmo c pressure while other cells cannot. This is due to specialized plant structures such as the cell wall which prevent lysis caused by osmosis. By taking advantage of this system, you will be able to look at the effects of tonicity in a biological system.

Materials
20% Sodium chloride (NaCl) solu on Several types of potatoes (e.g. russet, Yukon, yams)* 2 Plas c test tubes 100mL Graduated cylinder Water* 2 Pipe es

Knife for cu ng* Ruler Paper towel* Watch* Permanent marker *You must provide

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Lab 7: Osmosis
Procedure
1. Label two test tubes (A and B) for each type of potato you will be tes ng. Be sure to write the type of potato on the tube as well. Fill in the types of potato used in this experiment in the “Type of Potato” column of Table 3. 2. Carefully cut two strips of each type of potato on a cu ng board. The strips should be as close to 10cm long and 1cm wide as you can cut them so they fit into the test tube. Note: In the next step, you will account for any variability by measuring the volume of water displaced when submerged into a beaker containing a known volume of water. 3. Fill the 100mL graduated cylinder with 50mL of water. Place one strip of the first type of potato (Sample A) into the graduated cylinder and record the amount of water it displaced in the “Ini al Displacement” column of Table 3 in the row corresponding with the sample tested. Note: Displacement is a measurement of change and is calculated by subtrac ng the original volume (50ml) from the final volume that you read a er the potato is added to the 50mls of water. (e.g. 57mL—50mL = 7mL) 4. Remove Sample A from the graduated cylinder and, if any water was lost, fill the graduated cylinder up again with 50mL of water. Place Sample A into the corresponding test tube. 5. Place the second strip of the same type of potato (sample B) into the graduated cylinder and, again, record the amount of water it displaced in the “Ini al Displacement” column of Table 3 row corresponding with the sample tested. 6. Remove Sample B from the graduated cylinder and, if any water was lost, fill the graduated cylinder up again with 50mL of water. Place Sample B into the corresponding test tube. 7. Repeat Steps 3-6 for each type of potato you will be tes ng. 8. Using the plas c dropper, add water to each of the test tubes with the A samples in them un l the water covers the potato strip. In a similar manner, add the 20% Sodium Chloride (NaCl) solu on to each of the test tubes containing the B samples. Note: Make sure your test tubes are upright during the experiment. It may be useful to use the test tube rack provided. 9. A er an hour, drain the liquid from the test tubes containing your samples. 10. Fill the 100mL graduated cylinder with 50mL of water. Repeat Steps 3-6 for each sample and record the displacement in the “Final Displacement” column of Table 3 row corresponding with the sample tested. 11. Complete the last column of Table 3 by subtrac ng the ini al displacement from the final displacement.

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Lab 7: Osmosis
Table 3: Amount of Water Displaced by Potato Samples Before and A er Experiment

Type of Potato

Sample A B A B A B

Ini al Displacement (mL)

Final Displacement (mL)

Net Change= Final Displacement - Ini al Displacement

Ques ons
1. What is measured when looking at the net change in displacement of the potato samples?

2. Different types of potatoes have varying natural sugar concentra ons. Explain how this may influence the experiment.

3. Based on the data from this experiment, hypothesize which potato has the highest natural sugar concentra on. Explain your reasoning.

4. What was the texture and your observa on of the potato samples prior to the experiment? A er? Did it vary by type of potato?

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Lab 7: Osmosis
5. Would this experiment work with other plant cells? What about animal cells? Why or why not?

6. From what you know of tonicity, what can you say about the plant cells and the solu ons in the test tubes?

7. What do your results show about the concentra on of the cytoplasm in the potato cells at the start of the experiment?

8. If the potato is allowed to dehydrate by si ng in open air, would the potato cells be more likely to absorb more or less water? Explain.

9. Could this experiment be performed with other solu ons (i.e., sugar)? Why? Design an experiment to test the effect of different solu ons (be sure to include controls) on tonicity in plant cells. Note: Use something other than a potato in your experiment!

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Biological Processes

Lab 8 Respira on

84

Lab 8: Respira on

Concepts to explore:
• • • • Cellular energy Respira on Anaerobic respira on Aerobic respira on

Introduc on
ATP is the energy currency of the cell. It is produced through a process called respira on. The energy molecules (ATP) generated through respira on, are available to fuel the processes of the cell as needed. When ATP levels become too low a special protein signals the cell to begin respira on. As long as all the cri cal components for the reac on are available, this cycle provides a constant source of energy for the cell. Respira on harvests biological energy from fuel molecules, such as carbohydrates, and stores it as ATP. Together with oxygen, the cell converts carbohydrates to carbon dioxide, water and most importantly energy. Respira on is a controlled, mul step process which slowly releases the energy stored in glucose and converts it to ATP. If all of this energy from glucose were released at once, most would be lost as heat and light. Carbohydrates contain high energy bonds that, when broken, release electrons. The first stage, glycolysis, breaks carbohydrates (glucose) into pyruvate molecules. Though the bonds holding pyruvate together contain a great deal of poten al energy, this step yields li le energy.
Yeast has been used to make leavened bread for centuries. When yeast undergoes fermenta on, CO2 is trapped between gluten and causes the bread to rise. Ethanol, another byproduct of yeast fermenta on, generates the alcohol content in beer, and the CO2 provides effervescence. What ingredients must be present in order for this process to occur?

Glycolysis occurs with or without oxygen and takes place in the cytoplasm outside the mitochondria. Interes ngly, it is a pathway found in all living things. C6H12O6 + 6O2 glucose oxygen

6CO2 + 6H2O + energy carbon dioxide water

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Lab 8: Respira on
Membrane
Anaerobic
2 ATP

Aerobic in mitochondria

6 Carbon Compound

Glucose
4 ADP

Oxidation

4 ATP
3 Carbon Compound

+O2

Pyruvate
2 ATP
Homolactic fermentation

CO2 + H2O

Yeast fermentation

Alcohol + CO2

Lactic Acid

34 ADP

34 ATP

Cytosol

Mitochondrion

Figure 1: Aerobic Respira on

Aerobic respira on takes place in the mitochondria (a specialized organelle) of the cell and uses oxygen as the final electron acceptor in the electron transport pathway. Pyruvate is oxidized to generate energy. Special molecules shu le electrons to the ATP produc on site. Since oxygen has a very high affinity for electrons, aerobic respira on is the most efficient means of producing ATP (36 per reac on). Anaerobic respira on takes place in the cytoplasm of the cell and uses other, less efficient, molecules to transport electrons. If the final transfer molecule is organic (contains a carbon), the process is called fermenta on. Fermenta on is an anaerobic process that reduce regenerate NAD+ so that glycolysis can con nue. Because it cannot fully break down the glucose molecule, fermenta on is far less efficient than aerobic respira on, genera ng only two ATP molecules.

During physical ac vity, cells require more energy. As long as enough oxygen can be delivered to cells, aerobic respira on dominates. When energy consump on exceeds the oxygen supply, anaerobic respira on starts. Lac c acid is a byproduct, and is what causes muscle soreness a er a hard workout!

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Lab 8: Respira on
Experiment 1: Fermenta on by Yeast
Yeast cells produce ethanol and CO2 during fermenta on. We will measure the produc on of CO2 to determine the rate of anaerobic respira on in the presence of different carbohydrates. Note: Sucrose (a disaccharide) is made up of glucose and fructose. Glucose is a monosaccharide.

Materials
5 Respirometers (Figure 2) 1% Glucose solu on 1% Sucrose solu on Equal™, Splenda™, and sugar packets 1 Yeast packet 4 250mL Beakers

100mL Graduated cylinder Warm water* Pipe es Watch or mer* Permanent marker Ruler Measuring spoon
*You must provide

Figure 2: To make a respirometer, obtain two test tubes that fit into each other – one small plas c test tube and one large glass test tube for each respirometer.

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Lab 8: Respira on
Procedure
1. Completely fill the smallest tube with water and invert the larger tube over it. Push the small tube up (into the larger tube) un l the top connects with the bo om of the inverted tube. Invert the respirometer so that the larger tube is upright (there should be a small bubble at the top of the internal tube). Repeat this several mes as prac ce – strive for the smallest bubble possible. When you feel comfortable with this technique, empty the test tube and con nue with this experiment. 2. Mix 1/4 tsp. of yeast into 175mL of warm (40-43°C) water in a 250mL beaker. S r un l dissolved. Note: Make sure the yeast solu on is s rred before each test tube is filled. 3. Label both the big and small test tubes 1-5. 4. In a 250 ml beaker, mix the 1 gram packet of Equal™ with 100mL of water. In another 250 mL, mix the 1 gram packet of Splenda™ with 100mL of water. In another 250mL beaker, mix HALF of the 4 gram packet of sugar with 200mL of water. 5. Fill the smaller test tubes with 15mL solu on as follows:

Tube 5: 1% sugar solu on Note: A good prac ce in the lab is to rinse the graduated cylinder between each use. 6. Then, fill each tube to the top with the yeast solu on. 7. Slide the corresponding larger tube over the small tube and invert it as prac ced. This will mix the yeast and sugar solu ons. 8. Place respirometers in the test tube rack, and measure the ini al air space in the rounded bo om of the internal tube. Record these values in the Table 1. 9. Allow the test tubes to sit in a warm place (~37˚C) for one hour. A few sugges ons are: a sunny windowsill, atop (not in!) a warm oven heated to 200˚F, under a very bright (warm) light, etc.

• • • • •

Tube 1: 1% glucose solu on Tube 2: 1% sucrose solu on Tube 3: 1% Equal™ solu on Tube 4: 1% Splenda™ solu on

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Lab 8: Respira on
10. At the end of the respira on period, measure the air space in the internal tubes, and record in Table 1.
Table 1: Gas Produc on

Tube 1 2 3 4 5

Ini al gas height (mm)

Final gas height (mm)

Net Change

Ques ons
1. Hypothesize why some substances were not metabolized, while others were. Research the chemical formula of Equal™ and Splenda™ and explain how it would affect respira on.

2. If you have evidence of respira on, iden fy the gas that was produced. Suggest two methods for posi vely iden fying this gas.

3. How do the results of this experiment relate to the role yeast plays in baking?

4. What would you expect to see if the yeasts’ metabolism was slowed down? How could this be done?

5. Indicate sources of error and suggest improvement.

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Lab 8: Respira on
6. Op onal: Using the le over yeast, test some other carbohydrates (e.g. brown sugar, molasses) from your cupboard to see if the rates of respira on differ. Report your experimental procedure and results below.

Experiment 2: Aerobic Respira on in Beans
We will evaluate respira on in beans by comparing carbon dioxide produc on between germinated and non-germinated beans. As shown in the balanced equa on for cellular respira on, one of the byproducts is CO2 (carbon dioxide): C6H12O6 + 6H2O + 6O2 energy + 6CO2 +12H2O

We will use a carbon dioxide indicator ( bromothymol blue) to show oxygen is being consumed and carbon dioxide is being released by the beans. Bromothymol blue is an indicator that turns yellow in acidic condi ons, green when it is neutral and blue when it is in basic condi ons. When carbon dioxide dissolves in water, carbonic acid is formed by the reac on: H2O + CO2 H2CO3

resul ng in the forma on of this weak acid. If an indicator such as bromothymol blue is present, what do you think would happen? (Hint—what color would the indicator change to?)

Materials
100 Pinto beans 100 Kidney beans 6 250mL beakers Paper towels* 6 Measuring cups (small white paper cups) Pipe e

24mL Bromothymol blue solu on Parafilm 6 Rubber bands Water* *You must provide

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Lab 8: Respira on
Procedure
Figure 3: Beaker set-up

1. Fill two beakers with 200ml water. 2. Soak 50 pinto beans in Beaker 1P and 50 kidney beans in Beaker 1K for 24 hours. 3. Empty the water from beakers 1P and 1K. 4. Pour the soaked beans onto paper towels, keeping them separated. 5. Label the remaining beakers: Beakers 2P, 3P, Beaker 2K, and 3K. 6. Place several layers of moist paper towels at the bo om of the 250ml beakers. 4. Place 50 pre-soaked pinto beans into Beaker 1P, 50 control pinto beans in Beaker 2P, and zero beans in Beaker 3P. 5. Place 50 pre-soaked kidney beans into Beaker 1K, 50 control kidney beans in Beaker 2K, and zero beans in Beaker 3K. 6. Dispense 4ml of bromothymol blue solu on into each of the three measuring cups, and place the measuring cup inside each beaker (Figure 3). 7. Stretch Parafilm across the top of each beaker. Secure with a rubber band to create an airght seal. Note: If your Parafilm breaks, plas c wrap can also be used. 7. Place the beakers on a shelf or table, and let sit undisturbed at room temperature. 8. Observe the jars at 30 minute intervals for three hours, and record any color change of the bromothymol blue in Tables 2 and 3. 9. Let the beans and the jar sit overnight. Record your observa on in Tables 2 and 3.
Table 2: Bromothymol Blue Color Change Over Time for Pinto Bean Experiment

Time 0 min 30 min 60 min 90 min 120 min 150 min 180 min 24 hours

Beaker with pre-soaked

Beaker with un-soaked beans

Beaker with no beans

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Lab 8: Respira on
Table 3: Bromothymol Blue Color Change Over Time for Kidney Bean Experiment

Time 0 min 30 min 60 min 90 min 120 min 150 min 180 min 24 hours

Beaker with pre-soaked

Beaker with un-soaked beans

Beaker with no beans

Ques ons
1. What evidence do you have to prove cellular respira on occurred in beans? Explain.

2. Were there differences in the rates of respira on in pinto beans vs. kidney beans? If so, why?

3. If this experiment were conducted at 0°C, what difference would you see in the rate of respiraon? Why?

4. What is the mechanism driving the bromothymol blue solu on color change?

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Lab 8: Respira on
5. What are the controls in this experiment, and what variables do they eliminate? Why is it important to have a control for this experiment?

6. Would you expect to find CO2 in your breath? Why?

7. What effect would large changes of temperature (e.g., 37°C vs. 45°C) have on respira on in beans? Design an experiment to test your hypothesis, complete with controls.

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Biological Processes

Lab 9 Enzymes

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Lab 9: Enzymes

Concepts to explore:
• • • • • • Enzymes Selec vity Catalysts Ac va on energy Ac va on site Reac on rates

Concepts to explore:
• • Ac vators Inhibitors

Introduc on
Enzymes are specialized proteins that serve as biological catalysts to decrease the ac va on energy normally needed for a reac on to occur. This means the reac on rate is up to millions of mes faster than it would be without the enzyme. Most biochemical reac ons require enzymes for them to occur at fast enough rates to be useful. Typical nomenclature for enzymes follows the pa ern using the name of the substrate or the chemical reac on it catalyzes, and ends with “-ase”, e.g. catalase, amylase. (In other words, any me you see a word end in “ase” you know it is an enzyme).

Figure 1: The specificity of enzymes is controlled by their lock and key fit with a specific substrate.

Enzymes are extremely selec ve, and are o en described as having a “lock and key” fit (Figure 1). Their shape determines which substrates they bind and interact with. The ac va on site

97

Lab 9: Enzymes is the pocket where the substrate a aches and where the reac on occurs. A er the enzyme/substrate complex forms and catalysis occurs, the “new” substrate is released from the ac ve site, and the enzyme can repeat the process. Enzymes levels are not reduced or altered during the reac on. This means they are efficient and can be used repeatedly. Enzymes determine the rate at which the reac on occurs (not how it occurs). Their ac vity is affected by temperature, pH, enzyme and substrate concentra on, and other chemicals that may be present (such as salts, which can change the protein structure). Varia ons in temperature and alkalinity can change the shape of the proteins, such as enzymes, which makes them inac ve (they can no longer bind to their substrate). The pH can alter charge of the protein, once again changing its shape and rendering them inac ve. The concentra ons of both the enzyme and substrate determine the reac on rate (Figure 2). Remember that high reac on rates do not always translate into rapid me of comple on (it also depends on the amount of substrate!).

Figure 2: Substrate Satura on Curve

Ac vators are chemicals that bind to the ac ve site of the enzyme and help it to bind to the substrate. They are some mes called cofactors or organic coenzymes. Inhibitors are chemicals that interfere with the binding of the substrate to the enzyme. There are two types:

Many drugs and poisons are enzyme inhibitors. For example, aspirin inhibits an enzyme that leads to inflamma on.

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Lab 9: Enzymes
• •
Compe ve (can be replaced by the substrate) ve (not removed by the substrate)

Non-compe

Normal cellular processes produce toxic substances (waste) such as hydrogen peroxide and free radicals that if not eliminated, will kill the cell. Luckily, yeast and other organisms (including humans) have an enzyme called catalase that breaks down hydrogen peroxide into oxygen and water, both harmless to cells.

Experiment 1: Effect of Enzyme Concentra on
Yeast cells contain catalase. The effect of catalase can be seen when yeast is combined with hydrogen peroxide (Catalase: 2H2O2 ─› 2 H2O + O2). In this lab you will examine the effects of enzyme (catalase) concentra on based on the amount of oxygen produced.

Materials
Yeast Measuring Spoon 3 Test tubes 3 100mL Beakers Hydrogen peroxide 10 ml Graduated cylinder Permanent marker

Ruler String* 3 Balloons Watch* *You must provide

Procedure
1. Label three test tubes 1, 2, and 3 with a permanent marker. 2. Fill each tube with 10mL hydrogen peroxide. 3. Label three beaker A, B, C. 4. Add 1/2 teaspoon yeast (1 g) to 100 ml warm water (30-35°C) in Beaker A. Mix well by pipe ng.
Figure 3: When catalase is added to hydrogen peroxide, oxygen is released.

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Lab 9: Enzymes
5. Make a serial dilu on of yeast solu on by adding 10mL yeast solu on from Beaker A and transfer to Beaker B. Add 90 mL warm water (30-35°C) to Beaker B. Mix well by pipe ng. 6. Take 10mL yeast solu on from Beaker B and transfer to Beaker C. Add 90 mL warm water (3035°C) to Beaker C. Mix well by pipe ng. 7. Into the first test tube, add 5mL from Beaker A. 8. Quickly a ach a balloon to the top of the test tube so that it will fill with the oxygen produced by the enzyme reac on. It is important to execute this step quickly so that every bit of gas produced will be captured. 9. Repeat this procedure by transferring 5ml from Beaker B into test tube 2, a aching the balloon, and transferring 5 mL from Beaker C into test tube 3 and immediately placing the balloon on top. 10. Swirl each tube to mix, and wait 30 seconds.
11. Wrap the string around the center of the balloon to measure the circumference. Measure the

length of string with a ruler. Record measurements in Table 1 below.
12. Repeat step 11 for the remaining balloons. Table 1: Effect of Enzyme Concentra on on the Produc on of Gas

Tube 1 2 3

Amount of yeast 0.05g 0.005g 0.0005g

Balloon circumference (cm)

Ques ons
1. What is the enzyme in this experiment? What is the substrate?

2. Did you no ce a difference in the rate of reac on in the tubes with different concentra ons of enzymes? Why or why not?

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Lab 9: Enzymes
3. What was the effect of using less enzyme on your experiment?

4. Do you expect more enzyme ac vity if the substrate concentra on is increased or decreased? Draw a graph to illustrate this rela onship.

5. Hydrogen peroxide is toxic to cells, yet is a common byproduct of the reac ons that occur inside the body. How can this compound by changed to become non-toxic (Hint: Look at the chemical formula of hydrogen peroxide)?

Experiment 2: Effect of Temperature on Enzyme Ac vity
This experiment looks at the effect of temperature on enzyme ac vity.

Materials
Yeast Measuring spoon 3 Test tubes Hydrogen peroxide 10 mL Graduated cylinder 3 Balloons 2 Beakers

Hot water bath* Permanent marker Ruler String* Watch* Thermometer *You must provide

Procedure
1. With a permanent marker, label test tubes 1, 2, and 3. 2. Fill each tube with 10mL hydrogen peroxide. 3. Place tube 1 in the refrigerator, leave tube 2 at room temperature, and place tube 3 in the hot water bath (>85°C).

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Lab 9: Enzymes
4. Record the temperatures of each condi on in the table below. Let tubes sit for 15 minutes. 5. A er the elapsed me, remove tubes from the refrigerator and the boiling water bath. 6. Add 1/4 teaspoon of yeast to the refrigerated test tube. 7. Quickly a ach a balloon to the top of the test tube so that it will fill with the oxygen produced by the enzyme reac on. It is important to execute this step quickly so that every bit of gas produced will be captured. 8. Repeat steps 6-7 for the test tube in the hot water bath, then the room temperature test tube. 9. Swirl each tube to mix, and wait 30 seconds. 10. Wrap the string around the center of the balloon to measure the circumference. Measure the length of string with a ruler. Record measurements in the Table 2 below. 11. Repeat step 10 for the remaining balloons.
Table 2: Effect of Temperature on the Produc on of Gas

Tube Refrigerator Room temperature Hot water

Temperature ˚C

Balloon circumference (cm)

Ques ons
1. What is the enzyme in this experiment? What is the substrate?

2. How does temperature affect enzyme func on?

3. Do plants and animals have an enzyme that breaks down hydrogen peroxide? How could you test this?

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Lab 9: Enzymes
4. How can enzyme ac vity be increased?

5. Design an experiment to determine the op mal temperature for enzyme func on, complete with controls. Where would you find the enzymes for this experiment? What substrate would you use?

6. Draw a graph of balloon diameter vs. temperature. What is the correla on?

Experiment 3: Enzymes in Food
This experiment demonstrates the presence of amylase in some common foods. It is important to remember that amylase breaks down starch and iodine is a starch indicator that turns dark purple in the presence of starch.

Materials
Starch solu on Iodine-Potassium iodide (IKI) Empty 2 oz. bo le (i.e. corn syrup bo le) 2 Spray lids Permanent marker Paper towel*

Ginger root* 2 or more food products (e.g. apple, potato)* *You must provide

Note: Test as many foods as you like!

Procedure
1. A ach the spray lid to the starch solu on. Pour the remaining IKI solu on into an empty 2 oz. bo le (i.e. corn syrup bo le) and a ach a spray lid. Note: When pouring the IKI into the empty bo le, first, be sure the bo le has been thoroughly cleaned. Also, pour the IKI solu on into a graduated cylinder and from there pour it into the empty bo le to avoid any possible spills.

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Lab 9: Enzymes
2. Spray a paper towel with starch. Let dry for 2 hours. 3. In the mean me, set up a control for this experiment. (Hint: What happens when IKI solu on is mixed with starch? What happens when it is mixed with another liquid?) 4. Cut the food specimens so that a fresh surface is exposed. 5. Gently rub each specimen on the paper towel, back and forth 10-15 mes. Label where each specimen was rubbed on the paper towel with a permanent marker. 6. Rub a drop of saliva into the paper towel. (We know saliva contains amylase.) 7. Wait 5 minutes. 8. Hold the spray bo le 10-12 inches from the paper towel, and mist with the IKI solu on spray. 9. Observe where color develops, indica ng the presence of starch.

Ques ons
1. What is the func on of amylase? What does amylase do to starch?

2. What were your controls for this experiment? What did they prove?

3. Why did you wait 5 minutes before spraying with IKI solu on?

4. Which of the foods that you tested contained amylase? Which did not? What experimental evidence supports your claim?

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Lab 9: Enzymes
5. There is another diges ve enzyme other than salivary amylase that is secreted by the salivary glands. What is this enzyme? What substrate does it act on? Where in the body does it become ac vated, and why?

6. Saliva does not contain amylase un l babies are about two months old. How could this affect an infant’s diges on? (Hint: babies do not eat cereal un l about three months old)

7. Many common household products contain enzymes (e.g., meat tenderizer, enzyme detergent). Can you think of any other household uses for enzymes?

8. The stomach contains enzymes that aid in diges on, including proteases which digest proteins. Why don’t these enzymes digest the stomach and small intes ne, which are par ally composed of protein?

105

106

The Cell

Lab 10 Cell Structure and Func on
107

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Lab 10: Cell Structure & Func on

Concepts to explore:
• • • • • What is a cell? Prokaryotes Eukaryotes Cell structure Func on of cell structures

Introduc on
A cell is the fundamental unit of life. All living organisms originate from a single cell. Some remain as a single cell, while others become mul -cellular (like you!). Though most cells are difficult to see with the naked eye, using the microscope, cytologists have iden fied many of their features. These range from the characteris cs of the outer membranes, to internal structures such as the nucleus and mitochondria and have become the founda on for what is now known as “cell theory”. Cell theory states:

• • • • •

All cells are generated from previous cells All cells pass on their gene c informa on All living things are made of cell(s) Energy metabolism occurs inside cells The chemical make-up of cells is similar
Cytologists are scien sts who study cells. The study of the cell is known as cytology.

Although all organisms are made up of cells, not all cells are iden cal. Prokaryotes and eukaryotes are two structurally different types of cells.

• Prokaryotes are the most primi ve and basic organisms. They lack a membrane bound nucleus and membrane bound organelles (specialized structures). The term prokaryote comes from the La n words “pro” (before) and “karyote” (nucleus).

• Eukaryote are much more complex organisms, containing both a nucleus and membrane bound organelles. The term “eukaryote” comes from the La n words “eu” (true) and “karyote” (nucleus). Pro sts, fungi, plant and animal cells are all eukaryo c cell(s).

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Lab 10: Cell Structure & Func on
Prokaryotes
• Cyanobacteria and archaea (both primi ve bacteria) are the only prokaryotes.

Eukaryotes
• 2 billion years younger than prokaryo c cells

• Great biological diversity • All mul -cellular organisms are eukaryotes • Are very small (.1µm to 2µm) • Reproduce asexually. This means sexual reproduc on is absent, and there is li le gene c varia on between genera ons

• Significantly larger than most prokaryo c cells

• Have simple cellular components • Are capable of living almost anywhere and o en thrive in harsh condi ons

• More complex shapes and internal structure than prokaryotes

• Some are capable of capturing light energy (chloroplasts in plant cells and cones and rods of the eye)

• Are unicellular

Figure 1: A sample prokaryote

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Lab 10: Cell Structure & Func on
NOT SHOWN:

• Glycocalyx: A “slime coa ng” on some prokaryo c cells that helps protect the cell and enables it to a ach to “unconven onal” surfaces (i.e. teeth, lungs, ar ficial joints).

• Thylakoid: Extensions of the plasma membrane of cyanobacteria containing photosynthe c pigments.

• Flagella: Long cylindrical protrusions that rotate to provide mobility. • Pili: Hair like extensions on the cell surface that transmit gene c informa on and help secure the bacteria to its host. Eukaryotes:
Figure 2: Structures unique to eukaryotes

NOT SHOWN:

• Nucleolus: A part of the nucleus that is made of RNA, Protein and Chroma n and manufactures RNA and ribosomes.

• Cytoskeleton: The “skeleton” found in all eukaryo c cells that provides shape to the cell while also enabling it to move. It consists of three parts:

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Lab 10: Cell Structure & Func on
• Microfilaments: Small strands that help the cell resist tension. Think of it as a piece of wire.

• Intermediate filaments: Anchors the organelles in the cell and provide addi onal stability. • Microtubules: Small hollow tubes that help the cell maintain its shape, move things around within the cell and form other key structures.

• Centriole: Barrel shaped structures that help make cilia and flagella. They also play a key role in cell division.

• Cilia: Small “hairs” on the outside of the cell. They help the cell move and are sensory receptors.

• Flagella: The structure of eukaryo c flagella is far more complex than prokaryo c flagella. They provide mobility by rota ng back and forth, they help transport fluids and serve as sensory receptors.

• Mitochondria: The “power plant” of the cell. They are a membrane bound organelle (inner and outer membrane) with their own circular DNA, and make ATP (energy) for the rest of the cell.

• Chloroplast: Think of them as the plant version of mitochondria. The main difference is that they take light energy and convert it to mechanical energy.

• Peroxisomes: Contain enzymes that help the cell destroy toxins. • Vacuole: Membrane bound “sacs” that provide storage and provide transporta on within the cell (excre on, secre on).

• Vesicle: Plays a similar role to vacuoles, but are smaller.

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Lab 10: Cell Structure & Func on
Prokaryo c vs. Eukaryo c Cells
Structure Nucleus Plasma Membrane Cell Wall Cytoplasm Flagella and Pili Cilia Glycocalyx Cytoskeleton Endoplasmic Re culum Mitochondria Golgi Apparatus Chloroplast Ribosome Lysosome Peroxisome Vacuole and Vesicle Prokaryo c Cell No Yes Yes Yes Occasionally No Occasionally No No No No No Yes No No No Eukaryo c Cell Yes Yes Yes (in most cells) Yes Flagella- Occasionally Pili - No Occasionally Occasionally Yes Yes Yes Yes In plants and many pro sts Yes Yes Yes Yes (in most cells)

Experiment 1: Iden fying cell structures
View the slide pictures and images below, paying a en on to detail, and note the different characteriscs of prokaryotes and eukaryotes. On each picture, label the parts indicated if they are visible. If you can not see them, draw and label them where they would be located.

Figure 3

Bacteria: Nucleoid, cell wall, plasma membrane, ribosomes, glycocalyx (if present), flagella (if present)

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Lab 10: Cell Structure & Func on

Figure 4

Pro st: Nucleus, plasma membrane, cytoplasm, chloroplasts (if present), flagella (if present)

Figure 5

Plant Cell: Nucleus, cell wall, plasma membrane, cytoskeleton, cytoplasm, chloroplast, mitochondria, vacuoles

Figure 6

Animal Cell: Nucleus, nucleolus, nuclear envelope, plasma membrane, cytoplasm, mitochondria, golgi apparatus, ER (rough and smooth), ribosome, lysosomes, peroxisomes, vesicles 114

Lab 10: Cell Structure & Func on
Ques ons
1. For each structure iden fied, do you think its loca on affects its ability to func on? Why or why not? (Hint: those buried deep in the cell probably do different things than those closer to the cell membrane)

2.

Draw a labeled diagram of a small sec on of the plasma membrane and briefly describe its structure and func on.

3.

Describe the differences between animal and plant cells.

4.

Which of the following structures are present in both prokaryo c and eukaryo c cells? Plasma membrane, Golgi apparatus, DNA, lysosomes and peroxisomes, cytoplasm

5.

Where is gene c material found in plant cells?

6.

Mitochondria contain their own DNA (circular) and have a double membrane. What explanaon for this observa on can you come up with? (Hint 1: Where else do we see circular DNA?) (Hint 2: What do you know about the rela ve age of eukaryo c cells?)

7.

How is the structure of the cellulose wall related to its func on?

8.

Defects in structures of the cell can lead to many diseases. Pick one structure of a eukaryo c cell and develop a hypothesis as to what you think the implica ons would be if that structure did not func on properly.

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Lab 10: Cell Structure & Func on
9. Using books, ar cles, the internet, etc. conduct research to determine if your hypothesis was correct.

Experiment 2: Create a Cell
In this experiment you will create an animal and a plant cell using household items, to observe the difference between the two types of cells.

Materials
4 Unflavored gela n packets 2 Resealable bags Warm water* Tupperware* Bowl* Household items to use as cell structures* *You must provide

Procedure
1. Place four packets of unflavored gela n in a bowl . Prepare according to the direc ons on the package, but do not place it in the refrigerator. Locate household items that can serve as the nucleus, mitochondria, ribosomes, ER, golgi apparatus and chloroplasts.
For example, a plum works great as a nucleus, small mandarin oranges work great as mitochondria, string works well for the ER, etc. Be crea ve and come up with your own ideas!

2.

3.

Open one resealable bag (these serve as the cell membrane) and pour half of the liquid gela n into it. We will first make the plant cell so add the items that you have designated for each cell structure into the gela n and ghtly close the bag.

4.

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Lab 10: Cell Structure & Func on
5. 6. 7. Place the bag in the square disposable Tupperware (this serves as the plant cell wall). Open the other resealable bag and pour the remainder of the gela n into it. To make the animal cell, add the items you have designated for each cell structure into the bag and close it ghtly. Place both “cells” into the refrigerator for 24 hours. Return a er 24 hours and observe the “cells” you have made. No ce the difference between the animal cell and the plant cell.

8. 9.

Ques ons
1. What cell structures did you place in the plant cell that you did not place in the animal cell?

2.

Is there any difference in the structure of the two cells?

3.

What structures do organisms that lack cell walls have for support?

4.

How are organelles in a cell like organs in a human body?

5.

How does the structure of a cell suggest its func on? List three examples.

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118

The Cell

Lab 11 Mitosis
119

120

Lab 11: Mitosis

Concepts to explore:
• • • • • • Chromosomes Cell cycle Mitosis Interphase Metaphase Anaphase

Concepts to explore:
• • Telophase Cytokinesis

Introduc on

DNA is o en referred to as the “code of life”. Within the nucleus of each cell, it contains all the informa on necessary for that cell, or any mul cellular organism (you), to func on. It is a long, con nuous strand, ghtly packed and stored in large molecules called chromosomes (Figure 1).

If the DNA from a single cell was uncoiled and stretched out, it would be over 2 meters (6 feet) long, but too thin to see.

Mitosis is the process where soma c cells (non-sex cells) replicate and divide their nucleus. Before mitosis occurs (Figure 1), each cell has two complete sets of chromosomes (one set from your mother and one set from your father – “2n”). The process of mitosis generates diploid (2n) daughter cells that contain two complete sets of chromoFigure 1: Human karyotype somes iden cal to the parent cell. Mitosis is repeated every me a cell divides, trillions of mes in a human body. The life of a cell is divided into four stages, which are repeated un l the cell receives instruc ons to do otherwise. During this cell cycle, the cells duplicate their genomes, segregate that informa on, and divide, producing daughter cells. The stages in the cell cycle are:

•G1: the first growth phase during which the cell grows and makes the components necessary for replica on
Figure 2: The stages of the cell cycle

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Lab 11: Mitosis
•S: DNA replicates •G2: the second growth phase in which cellular organelles (mitochondria, ribosomes, and centrioles) are replicated.

•M: mito c division (prophase, metaphase, anaphase and telophase)
A cell normally completes the cycle in 18-24 hours, with mitosis occupying 1-2 hours of that me (Figure 2). Each stage is regulated by specialized proteins that coordinate the division and cell growth. Certain types of cancer are associated with the failure of these proteins.

Figure 3: Two chroma ds are joined together by a centromere to form a chromosome pair.

Chromosomes are joined as a four-arm structure by a centromere (Figure 3). During cell division (both meiosis and mitosis) each chromosome is duplicated, resul ng in two iden cal chroma ds. For a short while the cell contains four copies of each chromosome (two from your mother and two from your father). The chroma ds are then pulled apart and divided into daughter cells.

In mitosis, the division of the parent cell produces two cells, each having two sets of chromosomes (diploid (2n)). Thus, soma c cells can replicate and maintain the right number of chromosomes. Mitosis (Figure 4):

•

Interphase: The longest period of the cell cycle was named Interphase because it was the “in-between” period between cell cycles. It is the phase where the cell grows, its DNA replicates and it prepares for division. The replicated chromosomes pair up as sister chromads (exact copies of each other). Prophase: The nuclear membrane breaks down and the chromosomes separate. Structures which will serve as anchors in the cell (centrioles) during the division process appear. Metaphase: The chromosomes line up in the middle of the cell. Microtubules a ach to the chromosomes. The orienta on of each pair of homologous chromosomes is independent from all other chromosomes. This means they can “flip flop” as they line up, effec vely shuffling their gene c informa on into new combina ons. Microtubules (long strands) grow from each centriole and link together while also a aching to each pair of homologous chromosomes. Anaphase: The microtubules pull the sister chroma ds apart. Telophase: One set of chromosomes arrives at each centriole, at which me a nucleus

• •

• •

122

Lab 11: Mitosis forms around each set.

•

Cytokinesis: The plasma membrane of the cell folds in and encloses each nucleus into two new diploid daughter cells.

Figure 4: The stages of mitosis

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Lab 11: Mitosis
Experiment 1: Observa on of Mitosis in a Plant Cell
Mitosis is virtually iden cal in plant and animal cells, however, there is one small difference which occurs during telophase. Plants, due to the presence of a cell wall, can not “pinch” the cytoplasm into two daughter cells. Instead, a new cell wall must be developed which will then separate the two cells, allowing them both to be fully covered with a cell wall. In this experiment we will look at the different stage of mitosis in an onion cell. The large size of the onion cell allows the different stages of mitosis to be observed with the aid of a microscope. Also, when you are asked in the lab to specify the amount of me each stage takes during the cell cycle, remember that mitosis only occupies 1-2 hours while interphase can take anywhere from 18-24. With this informa on, you will be asked in the procedure below to calculate the percentage of cells in each stage of the cell cycle.

Materials
Prepared Allium root p digital slide picture (Figure 5)

Procedure
1. The length of the cell cycle in the onion root p is about 24 hours. Predict how many hours of the 24 hour cell cycle you think each step takes in Table 1. Examine a digital slide picture (following page) of an onion root p. Count the number of cells in each stage within a single field of view. Record this number in Table 1.

2.

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Lab 11: Mitosis
Figure 5: Onion root p slide

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Lab 11: Mitosis
Table 1: Number of Cells Observed in Each Stage of the Cell Cycle

Stage Interphase Prophase Metaphase Anaphase Telophase Totals 3.

Predicted %

Number of cells in stage

Total number of cells in field

Calculated %

Reexamine the onion root p slide at low magnifica on. Locate the region just above the root cap (use digital slide picture on previous page if you do not have the slide set). Focus on this zone of the onion root p and locate the stages of the cell cycle. Using the space below, draw the dividing cell in the appropriate area for each stage of the cell cycle.

4. 5.

Mitosis Interphase

Prophase

Prometaphase

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Lab 11: Mitosis
Metaphase

Anaphase

Telophase

Ques ons
1. What stage were most of the onion root p cells in? Does this make sense?

2. As a cell grows, what happens to its surface area : volume ra o? (Think of a balloon being blown up). How is this changing ra on related to cell division?

3. What is the func on of mitosis in a cell that is about to divide?

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Lab 11: Mitosis
4. How accurate were your me predic ons for each stage of the cell cycle?

5. Discuss one observa on that you found interes ng while looking at the onion root p cells.

6. What would happen if mitosis were uncontrolled?

128

The Cell

Lab 12 Meiosis
129

130

Lab 12: Meiosis

Concepts to explore:
• • • • Meiosis Diploid cells Haploid cells Chromosomal crossover

Concepts to explore:

Introduc on
Meiosis only occurs in organisms that reproduce sexually. The process generates haploid (1n) cells called gametes (sperm cells in males and egg cells in females), or spores in some plants, fungi, and pro sts, that There are over two meters of DNA packcontain one complete set of chromosomes. Haploid cells aged into a cell’s nucleus. It is coiled and fuse together during fer liza on to form a diploid cell with folded into superhelices that form chrotwo copies of each chromosome (2n). mosomes, which must be duplicated beGenes are the units of heredity that have specific loci (loca ons) on the DNA strand and code for inheritable traits (such as hair color). Alleles are alterna ve forms of the same gene (brown vs. blue eyes). Homologous chromosomes contain the same genes as each other but o en different alleles. Non-sex cells (e.g. bone, heart, skin, liver) contain two alleles (2n), one from the sperm and the other from the egg. Mitosis and meiosis are similar in many ways. Meiosis, however, has two rounds of division—meiosis I and meiosis II. There is no replica on of the DNA between meiosis I and II. Thus in meiosis, the parent cell produces four daughter cells, each with just a single set of chromosomes (1n). Meiosis I is the reduc on division– the homologous pairs of chromosomes are separated so that each daughter cell will receive just one set of chromosomes. During meiosis II, sister chroma ds are separated (as in mitosis). fore a cell divides.

Each of the 23 human chromosomes has two copies. For each chromosome, there is a 50:50 chance as to which copy each gamete receives. That translates to over 8 million possible combina ons!

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Lab 12: Meiosis
Meiosis:
•

Prophase I: The sister chroma ds a ach to their homologous counterparts (same chromosome – different version). This is the stage where crossing over occurs (homologous chromosomes exchange regions of DNA). Structures which will serve as anchors in the cell (centrioles) during the division process appear. Metaphase I: The chromosomes line up in the middle of the cell. The orienta on of each pair of homologous chromosomes is independent from all other chromosomes. This means they can “flip flop” as they line up, effec vely shuffling their gene c informa on into new combina ons. Microtubules (long strands) grow from each centriole and link them together while also a aching to each pair of homologous chromosomes. Anaphase I: The microtubules pull the homologous chromosomes apart (the sister chroma ds remain paired). Telophase I: One set of paired chromosomes arrives at each centriole, at which me a nucleus forms around each set. Cytokinesis: The plasma membrane of the cell folds in and encloses each nucleus into two new daughter cells. Prophase II: Before any replica on of the chromosomes can take place, the daughter cells immediately enter into prophase II. New spindle fibers form as the nucleus breaks down. Metaphase II: The sister chroma ds align in the center of the cell, while the microtubules join the centrioles and a ach to the chromosomes. Unlike metaphase I, since each pair of sister chroma ds is iden cal, their orienta on as they align does not ma er. Anaphase II: The sister chroma ds are separated as the microtubules pull them apart. Telophase II: The chroma ds arrive at each pole, at which me a nucleus forms around each. Cytokinesis: The plasma membrane of the cell folds in and engulfs each nucleus into two new haploid daughter cells.

•

•

•

•

•

•

• •

•

We briefly discussed “crossing over” in Prophase I. Since the chromosomes of each parent undergoes gene c recombina on, each gamete (and thus each zygote) acquires a unique gene c fingerprint. The closeness of the chroma ds during prophase I, creates the opportunity to exchange gene c material (chromosomal crossover) at a site called the chiasma. The chroma ds trade alleles for all genes located on the arm that has crossed. The process of meiosis is complex and highly regulated. There are a series of checkpoints that a cell must pass before the next phase of meiosis will begin. This ensures any mutated cells are iden fied

132

Lab 12: Meiosis and repaired before the cell division process can con nue.

One of the muta ons that is of par cular concern is a varia on in the amount of gene c material in a cell. It is cri cal that the gamete contain only half of the chromosomes of the parent cell. Otherwise the amount of DNA would double with each new genera on. This is the key feature of meiosis.

Muta ons that are not caught by the cell’s self-check system can result in chromosomal abnormali es like Down’s syndrome, in which there are 3 copies of chromosome 21.

Figure 1: The stages of meiosis

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Lab 12: Meiosis
Experiment 1: Following Chromosomal DNA Movement
Every cell in the human body has two alleles that condense into single chromosomes held together by a centromere. These “sister” chroma ds replicate and pair with the newly made homologous chromosomes. In this exercise we will follow the movement of the chromosomes through meiosis I and II to create haploid (gamete) cells.

Materials
2 sets of different colored snap beads (32 of each) 8 centromeres (snap beads) Blue and red markers* *You must provide

Figure 2: Bead Set-up

Procedure
Meiosis I A. As prophase I begins, chromosomes coil and condense in prepara on for replica on.

1. Using one single color of bead, build a homologous pair of duplicated chromosomes. Each chromosome will have 10 beads with a different colored centromere in it. For example, if there are 20 red beads, 10 beads would be snapped together to make two different strands. In the middle of each of the 10 bead strands, snap a different colored bead in to act as the centromere. Now, repeat these steps using the other color of bead. 2. Assemble another homologous pair of chromosomes using only 12 (that’s 6 per strand) of the first color bead. Place another, different colored bead in the middle of each to act is its centromere. Repeat this step (2 strands of 6 beads plus a centro-

134

Lab 12: Meiosis mere) with the other color of beads. B. Bring the centromeres of two units of the same color and length together so they can be held together to appear as a duplicated chromosome. 1. Simulate crossing over. Bring the two homologues pairs together (that’d be the two pairs that both have 10 bead strands) and exchange an equal number of beads between the two. C. Configure the chromosomes as they would appear in each of the stages of meiosis I.

Meiosis II A. Configure the chromosomes as they would appear in each stage of meiosis II. B. Return your beads to their original star ng posi on and simulate crossing over. Track how this changes the ul mate outcome as you then go through the stages of meiosis I and II. C. Using the space below, and using blue and red markers, draw a diagram of your beads in each stage. Beside your picture, write the number of chromosomes present in each cell. Meiosis I Prophase I

Metaphase I

Anaphase I

Telophase I

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Lab 12: Meiosis
Meiosis II Prophase II

Metaphase II

Anaphase II

Telophase II

Ques ons
1. What is the state of the DNA at the end of meiosis I? What about at the end of meiosis II?

2.

Why are chromosomes important?

3.

How are Meiosis I and Meiosis II different?

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Lab 12: Meiosis
4. Name two ways meiosis contributes to gene c recombina on.

5.

Why do you use non-sister chroma ds to demonstrate crossing over?

6.

How many chromosomes were present when meiosis I started?

7.

Why is it necessary to reduce the chromosome number of gametes, but not other cells of an organism?

8.

If humans have 46 chromosomes in each of their body cells, determine how many chromosomes you would expect to find in the following: Sperm ___________________ Egg ___________________ Daughter cell from mitosis ___________________ Daughter cell from Meiosis II ___________________

9.

Inves gate a disease that is caused by chromosomal muta ons. When does the muta on occur? What chromosome is affected? What are the consequences?

137

138

The Cell

Lab 13 DNA & RNA
139

140

Lab 13: DNA & RNA

Concepts to explore:
• • • • • • DNA structure Nucleo des Amino acids Proteins Gene c code Muta on

Concepts to explore:
• • • RNA Transcrip on Transla on

Introduc on
Long before we had any understanding of how, we knew that traits were passed on from genera on to genera on. We knew traits were expressed as heritable proteins, but we had no idea of the mechanism. Whatever the mechanism, it needed to meet three criteria: It needed to carry informa on between genera ons. It needed to express that informa on. It needed to be easily replicable. Prior to the 1950’s, there was much debate over what the structure of a molecule that met all three criteria would look like. Though a number of people made significant contribu ons, in 1953 James Watson and Francis Crick won the Nobel Prize for their model of what we now know as DNA (deoxyribonucleic acid). The features of this model sa sfied all of the necessary criteria.

• • •

Figure 1: DNA Double Helix

DNA takes the form of what is commonly referred to as a “double helix”, or perhaps more simply, a long twisted ladder with rungs (Figure 1). The sides of the ladder consist of a sugar-phosphate “backbone” and the strand itself has direc onality. In other words, like the words on this page, there is a set order in which they are read. In the case of DNA, it is from the 5’ (five prime) to 3’ (three prime) end. The rungs of the ladder carry informa on in a sequen al series of four different nucleo des (small molecules): Guanine (G), Adenine (A), Thymine (T) and Cytosine (C). These nucleo des pair up in a very precise manner (specificity); A with T, and G with C (Figure 2). No other combina ons are ever made because of the chemical and electrical forces within the nucleo de.
Figure 2: Nucleo des

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Lab 13: DNA & RNA
As a cell divides, the DNA double helix splits into a single helix (Figure 3). Each single helix then serves as a template for a new strand. Neighboring nucleo des then bind to the single strand helix a er which a new sugarphosphate backbone is formed. The specificity in which the nucleo des pair means the two new double helices (DNA) are iden cal to the original. It is the sequence of these nucleo des that are passed on from one genera on to another, as heritable informa on.

Figure 3: DNA replica on

So the ques on remains, why is the sequence of these different four nucleo des so important? Simply put, they instruct your cells what proteins to make and how to make them (your body is made of proteins). If the protein is wrong you are likely either very sick or dead. Proteins are simply chains of amino acids (small molecular building blocks) that are linked together. Twenty different amino acids are available to produce all the proteins in the body. Each amino acid is coded for by a three nucleo de sequence (codon). The sequence of the amino acids determines the size of the protein and how it will fold, both factors that determine its func on. Other factors such as charge and hydrophobicity (an aversion to water molecules) play a role in determining how a protein folds. Consider the following analogy: “The earth revolves around the sun.”

• Each of the 12 different le ers (codons) in the preceding sentence is largely uninforma ve. • When le ers are assembled they create words, which have meaning. • Linked words create a sentence (protein), which is then informa ve.
Consider a protein that is five amino acids long. Picking from the 20 available amino acids there are 520 different possible combina ons (3,200,000). Even small proteins are typically several hundred amino acids long. The number of different proteins that can ul mately be coded for by 20 amino acids is virtually limitless. The next obvious ques on is how do the 4 nucleo des “code” for 20 different amino acids. Each “le er” (codon) in the gene c code is made up of three nucleo des which codes for a specific amino acid. If we start with four possible nucleo des (A, T, G, C), how does your body make twenty different amino acids?

• If the “le er” is two nucleo des long, there are 16 possible “le ers” (24) - not enough. • If the “le er” is three nucleo des long, there are 64 possible combina ons (34) - more than what’s needed for twenty amino acids.

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Lab 13: DNA & RNA
Like a sentence, the reader (a cell) needs to know where to start and where to stop (two more codons, for a total of 22). The remaining 42 possible combina ons make up what is referred to as “the redundancy of the code”. In other words; Tim, Tom, Tam would all be the same person, it is simply three different spellings for his name. Each combina on of three nucleo de is known as a “codon”.

Experiment 1: Coding

Materials
For the following exercises: Red beads Blue beads Yellow beads Green beads

• •

Regular beads are used as nucleo des Pop-it beads are used as amino acids

Procedure
A) Using red, blue, yellow and green beads, devise and lay out a three color code for each of the following le ers (codon). For example Z = green : red : green. In the spaces below the le er, record your “code”.

C: ___ M: ___

E: ___ O: ___

H: ___ S: ___

I:

K: ___ T: ___ Stop: ___

L: ___ U: ___ Space: ___ ___

Create codons for: Start: ___

B) Using this code, align the beads corresponding to the appropriate le er to write the following sentence (don’t forget start, space and stop): The mouse likes most cheese 1. How many beads did you use?

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Lab 13: DNA & RNA
There are mul ple ways your cells can read a sequence of DNA and build slightly different proteins from the same strand. We will not go through the process here, but as an illustra on of this “alternate splicing”, remove codons (beads) 52-66 from your sentence above. 2. What does the sentence say now? (re-read the en re sentence)

Muta ons are simply changes in the sequence of nucleo des. There are three ways this occurs: Change one, remove one, or add one

Using the sentence from exercise 1B: C) Change the 24th bead to a different color. 3. What does the sentence say now? (re-read the en re sentence)

4. Does it make sense?

D) Replace the 24th bead and remove the 20th bead (remember what was there). 5. What does the sentence say (re-read the en re sentence)?

6. Does it make sense?

7. Where does it make sense?

E) Replace the 20th bead and add one between bead numbers 50 and 51.

8. What does the sentence say?

9. Does it make sense?

10. In “C” we mutated one le er. What role do you think the redundancy of the gene c code plays, in light of this change?

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Lab 13: DNA & RNA
11. Based on your observa ons, why do you suppose the muta ons we made in “D” and “E” are called frame shi muta ons.

12. Which muta ons do you suspect have the greatest consequence? Why?

DNA codes for all of the proteins manufactured by any organism (including you!). It is valuable, highly informa ve and securely protected in the nucleus of every cell. Consider the following analogy: An architect spends months or years designing a building. His original drawings are valuable and informa ve. He will not provide the original copy to everyone involved in construc ng the building. Instead, he gives the electrician a copy with the informa on he needs to build the electrical system. He will do the same for the plumbers, the framers, the roofers and everyone else who needs to play a role to build the structure. These are subsets of the informa on contained in the original copy. Your cell does the same thing. The “original drawings” are contained in your DNA which is securely stored in the nucleus. Nuclear DNA is “opened up” by an enzyme (Helicase) and a subset of informa on is transcribed (copied) into RNA. RNA is a single strand version of DNA, where the nucleo de uracil, replaces thymine. The copies are sent from the nucleus to the cytoplasm in the form of mRNA (messenger RNA). Once in the cytoplasm, tRNA (transfer RNA) links to the codons and aligns the proper amino acids, based on the mRNA sequence. The ribosomes (protein builders) that float around in the cytoplasm, latch onto the strand of mRNA and sequen ally link the amino acids together that the tRNA has lined up for them. This construc on of proteins from the mRNA is known as transla on.

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Lab 13: DNA & RNA

Experiment 2: Transcrip on and Transla on

Materials
Red beads Blue beads Green beads Yellow beads Pop-it beads (8 different colors)

Procedure
A) B) C) Write a five word sentence using no more than 8 different le ers. Using the four colored beads, create “codons” (3 beads) for each le er in your sentence, plus ones for “start, “space” and stop”. “Write” the sentence using the beads. 1. How many beads did you use?

Using your pop-it beads, assign one bead for each codon (you do not need beads for start, stop and space). These will be your amino acids. Connect the “Pop it” beads to build the chain of amino acids that codes for your sentence (leave out the “start”, “stop” and “space”). 2. How many different amino acids did you use?

3. How many total amino acids did you use?

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Lab 13: DNA & RNA
Experiment 3: DNA Extrac on

Materials
Fresh so fruit (i.e. grapes, berries or banana)* Cheesecloth Rubber band Plas c zipper bag 2 100mL Beaker DNA extrac on solu on**

Standing test tube Scissors* Ethanol (ice cold)*** S r s ck Rubber band *You must provide

** Sodium chloride, detergent and water ***For ice cold ethanol, store in the freezer 60 minutes before use.

Figure 4: DNA extracted from fruit was dyed with a substance that glows under black light.

Procedure
1. Put pieces of so fruit (approximate size of 5 grapes) into a plas c zipper bag and mash with your fist. 2. Using a 100ml beaker, measure out 10ml of the DNA extrac on solu on and pour it into the bag with the fruit it in. Seal the bag completely. 3. Mix well by kneading the bag for 2 minutes. 4. Create a filter by placing the center of the cheesecloth over the mouth of the standing test tube, pushing it into the tube about 2 inches, and securing the cheesecloth with a rubber band around the top of the test tube. 5. Cut a hole in the corner of the bag and filter your extrac on by pouring it into the cheese-

147

Lab 13: DNA & RNA cloth (the filtered solu on in the standing test tube is what you keep). 6. While holding the test tube at a 45° angle, slowly pour 5ml ice-cold ethanol into the test tube. 7. DNA will precipitate (come out of solu on) a er the ethanol has been added to the soluon. Let the test tube sit for 2 - 5 minutes. You should begin to see air bubbles form at the boundary line between the ethanol and the filtered fruit solu on. A er enough bubbles form you will see the DNA float to the top of the ethanol. 8. Gently insert the s r s ck into the test tube and slowly raise and lower the p several mes to spool and collect the DNA. If there is an insufficient amount of DNA available, it may not float to the top of the solu on in a form that can be easily spooled or removed from the tube. However, the DNA will s ll be visible as white/clear clusters by gently s rring the solu on and pushing the clusters around the top.

Ques ons
1. Which DNA bases pair with each other?

2. How is informa on to make proteins passed on through genera ons?

3. Why did we use a salt in the extrac on solu on?

4. What else might be in the ethanol/aqueous interface? How could you eliminate this?

5. What is the texture and consistency of DNA?

6. Is the DNA soluble in the aqueous solu on or alcohol?

7. What surprised you about DNA replica on and protein synthesis?

148

The Cell

Lab 14 Mendelian Gene cs
149

150

Lab 14: Mendelian Gene cs

Concepts to explore:
• • • • • • Gregor Mendel Law of segrega on Homozygous Heterozygous Independent assortment Dominant vs. recessive

• • • • • • •

Incomplete dominance Co-dominance Genotype Phenotype Monohybrid cross Dihybrid cross Punne square

Introduc on
In 1866, Gregor Mendel, an Austrian Monk, published a paper en tled “Experiments in plant hybridizaon”. It went largely unno ced un l 1900 when it was rediscovered and subsequently became the basis for what we now refer to as Mendelian Gene cs. Mendel was the first to recognize:

• •

Inherited characters are determined by specific factors (now recognized these as genes). These factors occur in pairs (genes).

When both alleles of a gene are the same they are said to be homozygous, while if they are different they are said to be heterozygous. When gametes form, these factors segregate so that each gamete contains only one allele for each gene. Remember, alleles reside on the chromosomes that are dividing. These original observa ons lead to what we now refer to as The law of segrega on and the law of independent assortment.

Figure 1: Law of Segrega on

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Lab 14: Mendelian Gene cs
The law of segrega on states that during meiosis, homologous (paired) chromosomes split (Figure 1). The law of independent assortment states that during meiosis, each homologous chromosome has an equal chance of ending up in either gamete, and alleles for individual genes segregate with the chromosomes on which they are located (Figure 2). Using corn as an example (Figure 2):
Figure 2: Law of Independent Assortment

• •

The large chromosome has the gene for kernel color (Y = yellow, y = blue). The small chromosome has the gene for kernel texture (S = smooth (green); s = wrinkled (red)).

When a dominant allele is present, that characteris c is expressed, regardless of the second allele. In this case both the Yy and YY offspring will be yellow. A recessive allele is only expressed when both alleles are recessive. In this case only the yy combinaon is blue. The dominant allele is always represented by capital le ers, while the recessive is represented by lower case le ers. Genotype refers to the combina on of alleles for a par cular trait. Phenotype refers to the appearance of that combina on of alleles. In our example, the genotype of the diploid cell is Yy, Ss, while the phenotype is Yellow and Smooth.

Figure 3: Monohybrid Cross Punne Square F1

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Lab 14: Mendelian Gene cs

Parent 1

Y
Parent 2

Y
Parent 2

Y Y YY

Parent 1

y Yy

y y

Yy Yy

Yy Yy

y

Yy

yy

Figure 4: Punnet square of a monohybrid cross (F1)

Figure 5: Punnet square of a monohybrid cross (F2)

Ys

YS

yS

ys

Ys

YY ss

YY Ss

yY Ss

yY ss

YS yS ys

YY sS Yy sS Yy ss

YY SS Y y SS Yy Ss

yY SS yy SS yy Ss

yY sS yy sS yy ss

Figure 6: Punnet square of a dihybrid cross (F1)

Alleles can exhibit incomplete dominance and co-dominance. An example of incomplete dominance is the cross of two plants, one with red flowers and one with white, whose offspring have pink flowers. In the case of codominance, the same cross would result in red and white striped flowers. If we know the genotype of two parents we can predict both the genotype and phenotype of their offspring using a Punne Square. A monohybrid cross is a cross between two parents (P), looking at a single gene (Figure 3). In this example, both parents are pure breeding (homozygous); one for the yellow color and one for the blue color. This cross can be shown as a Punne Square (Figure 4), with each parent (P) contribu ng a single gamete. The offspring (F1) are determined adding the gamete of each parent (P) (Row and Column). The F1 genotypes are all Y , y; with yellow phenotypes. The cross of the (F1) genera on, known as the F2 genera on, is shown in Figure 5. The Punne square can also predict the F1 for mul ple genes.

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Lab 14: Mendelian Gene cs
Using our corn example, let’s look at two genes (color and texture), also known as a dihybrid cross. In this example we use two parents that are heterozygous for both traits (Figure 6), using the gametes we already iden fied in (Figure 2). The F2 phenotypes are:

• • • •

Yellow & Smooth: Yellow & Wrinkled: Blue & Smooth: Blue & Wrinkled:

9 3 3 1

NOTE: 9: 3: 3: 1 Is a ra o you should remember for ques on #4

Experiment 1: Punne Square Crosses

Materials
Red beads Blue beads Green beads Yellow beads 2 100mL Beakers

Procedure
1. Set up and complete Punne squares for each of the following crosses: (remember Y = yellow, and y = blue) Y Y and Y y Y Y and y y

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Lab 14: Mendelian Gene cs
a) What are the resul ng phenotypes?

b) Are there any blue kernels? How can you tell?

2. Set up and complete a Punne squares for a cross of two of the F1 from 1b above: a) What are the genotypes of the F2 genera on?

b) What are their phenotypes?

c) Are there more or less blue kernels than in the F1 genera on?

3. Iden fy the four possible gametes produced by the following individuals: a) YY Ss: b) Yy Ss: ______ ______ ______ ______ ______ ______ ______ ______

c) Create a Punne square using these gametes as P and determine the genotypes of the F1:

d) What are the phenotypes? What is the ra o of those phenotypes?

4. You have been provided with 4 bags of different colored beads. Pour 50 of the blue beads and yellow beads into beaker #1 and mix them around. Pour 50 of the red beads and green beads in beaker #2 and mix them.

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Lab 14: Mendelian Gene cs

Attention! Do not pour the beakers together.

• • • • •

#1 contains beads that are either yellow or blue. #2 contains beads that are either green or red. Both contain approximately the same number of each colored bead. These colors correspond to the following traits (remember that Y/y is for kernel color and S/s is for smooth/wrinkled): Yellow (Y) vs. Blue (y) Green (S) vs. Red (s).

A. Monohybrid Cross: Randomly (without looking) take 2 beads out of #1.

• •

This is the genotype of individual #1, record this informa on. Do not put these beads back into the beaker. Repeat this for individual #2. These two genotypes are your parents for the next genera on. Set up a Punne square and determine the genotypes and phenotypes for this cross. Repeat this process 4 mes (5 total). Put the beads back in their respec ve beakers when finished.

•

a) How much genotypic varia on do you find in the randomly picked parents of your crosses?

b) How much in the offspring?

c) How much phenotypic varia on?

d) Is the ra o of observed phenotypes the same as the ra o of predicted phenotypes? Why or why not?

e) Pool all of the offspring from your 5 replicates. How much phenotypic varia on do

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Lab 14: Mendelian Gene cs you find?

f)

What is the difference between genes and alleles?

g) How might protein synthesis execute differently if there a muta on occurs?

h) Organisms heterozygous for a recessive trait are o en called carriers of that trait. What does that mean?

i)

In peas, green pods (G) are dominant over yellow pods. If a homozygous dominant plant is crossed with a homozygous recessive plant, what will be the phenotype of the F1 genera on? If two plants from the F1 genera on are crossed, what will the phenotype of their offspring be?

B. Dihybrid Cross: Randomly (without looking) take 2 beads out of beaker #1 AND 2 beads out of beaker #2.

• •

These four beads represent the genotype of individual #1, record this informa on. Repeat this process to obtain the genotype of individual #2. a) What are their phenotypes?

b) What is the genotype of the gametes they can produce?

•

Set up a Punne square and determine the genotypes and phenotypes for this cross.

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c) What is your predicted ra o of genotypes? Hint: think back to our example dihybrid cross

•

Repeat this process 4 mes (for a total of 5 trials). d) How similar are the observed phenotypes in each replicate?

e) How similar are they if you pool your data from each of the 5 replicates? f) Is it closer or further from your predic on?

g) Did the results from the monohybrid or dihybrid cross most closely match your predicted ra o of phenotypes?

h) Based on these results; what would you expect if you were looking at a cross of 5, 10, 20 independently sorted genes?

i)

Why is it so expensive to produce a hybrid plant seed?

j)

In certain bacteria, an oval shape (S) is dominant over round and thick cell walls (T) are dominant over thin. Show a cross between a heterozygous oval, thick cell walled bacteria with a round, thin cell walled bacteria. What are the phenotype of the F1 and F2 offspring?

5. The law of independent assortment allows for gene c recombina on. The following equa on can be used to determine the total number of possible genotype combina ons for any par cular number of genes: 2 = Number of possible genotype combina ons (where “g” is the number of genes) 1 gene: 2 genes: 2 = 2 genotypes 2 = 4 genotypes
2 1 g

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3 genes: 23 = 8 genotypes

Consider the following genotype: Yy Ss Tt We have now added the gene for height: Tall (T) or Short (t). a) How many different gamete combina ons can be produced?

b) Many traits (phenotypes), like eye color, are controlled by mul ple genes. If eye color were controlled by the number of genes indicated below, how many possible genotype combina ons would there be? 5: 10: 20:

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The Cell

Lab 15 Popula on Gene cs
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Lab 15: Popula on Gene cs

Concepts to explore:
• • • • • Gene pool Gene frequency Gene c varia on Gene c dri Founder effect

Concepts to explore:
• • Muta on Natural selec on

Introduc on
In the previous lab we looked at how genes are passed on to offspring. In this lab, the exercises are designed to look at individual genes (two alleles, one dominant, one recessive). However, we will be looking at their presence, prevalence and distribu on at the popula on level. The gene pool is the sum of all genes and their corresponding alleles in a given popula on. Take a look at the popula on of 100 brown and white mice in Figure 1. The color brown (B) is dominant. The standard for naming alleles is to use the case of the dominant trait, with the lower case to represent the recessive allele. Their gene pool is B, b. Gene frequency refers to how many mes each allele is found in the popula on. These 100 mice have 200 genes:
Figure 1: Mouse Population

• • •

55 heterozygous mice (B, b) have 55 B alleles and 55 b alleles. 27 homozygous recessive mice (b, b) have 54 b alleles (2 x 27 “b”= 54). 18 homozygous dominant mice (B, B) have 36 B alleles (18 x 2 “B” = 36).

The gene frequency of the popula on is: B: 91 b: 109

O en this is represented as a percentage of the dominant gene, in this case, the percentage of B is

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Lab 15: Popula on Gene cs
45.5% (=91/200). Note that the dominant gene is less prevalent than the recessive gene. This is not unusual, it is important to remember that dominance has no direct rela onship to prevalence.

Ques ons
1. What is the gene pool of this popula on?

2. What is the gene frequency?

Gene c varia on is simply the gene c difference within or between popula ons, in the gene pool and/ or gene frequency. Consider the following two popula ons of bu erflies:

Fact: Both popula ons contain the same 4 colors of bu erflies, thus the gene pool is the same. However, the distribu on of colors within that popula on is different, thus their gene frequencies are different.

NOTE: In these exercises on gene pool, gene frequency and gene c diversity; assume there are 4 alleles for color and that all bu erflies are homozygous.

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Lab 15: Popula on Gene cs
Experiment 1: Gene c Varia on

Materials
Blue beads Red beads Green beads Yellow beads 2 100mL Beakers 2 250mL Beakers

NOTE: When done return beads to their respec ve beakers (1 or 2).

Procedure
1. Pour 50 blue beads and 50 red beads into a 250 ml beaker. Without looking, randomly take 50 beads from the 250 ml beaker and place them in a 100 ml beaker (this is beaker #1). 2. Pour 50 green beads and 50 yellow beads into a second 250 ml beaker. Without looking, randomly take 20 beads from the 250 ml beaker and place them in the other 100 ml beaker (this is beaker #2).

Ques ons
1. What is the gene pool of beaker #1?

2. What is the gene pool of beaker #2?

3. What is the gene frequency of beaker #1?

4. What is the gene frequency of beaker #2?

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5. What can you say about the gene c varia on between these popula ons?

Gene c dri , the varia on of the gene pool and/or gene frequency of a popula on, can result from a variety of stochas c (random) means. Consider the following popula on of bu erflies who have half of their habitat destroyed by wildfire:

The remaining popula on has 50% of the ini al gene pool (2 colors) and the gene frequency is different. As these individuals reproduce, their offspring will no longer reflect the original popula on.

Experiment 2: Gene c dri

Materials
Blue Beads Red Beads Green Beads Yellow Beads Beads in beaker #1 Beads in beaker #2 1 100 mL beaker 1 250 mL beaker

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Lab 15: Popula on Gene cs

Procedure
1. From the 250 ml beaker containing green and yellow beads, take 10 beads and place them into the unused 100 ml beaker (this is beaker #3). 2. Remove half of the beads from beakers #1, #2, and #3 (keep them separated so they can be returned to the proper beaker). These are the individuals that survived the fire. 3. Record your results and place the beads back in their respec ve beakers. 4. Repeat this process 4 more mes (5 total).

Ques ons
1. What observa ons can you make regarding the gene pool and gene frequency of the surviving individuals?

2. What determines how o en a phenotype occurs in a popula on?

3. Are dominant characteris cs more frequent in a popula on than recessive characteris cs? Why or why not?

4. If a selec on pressure was against the trait of the dominant allele, what change in a popula on would you expect to see?

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Experiment 3: Founder Effect
The same popula on of bu erflies is in the path of a hurricane. All survive, but 10 are blown to a new loca on. These 10 start a new popula on, their progeny will reflect the founders gene pool. This is known as the founder effect.

Materials
Beaker #1 Beaker #2 Beaker #3

Note: When you are finished with this experiment, return the beads to their appropriate bags.

Procedure
1. Remove 10 individuals from beaker #1, 5 from beaker #2 and 2 from beaker #3. These are the founders of your new popula on. 2. Record your results and place the beads back in their respec ve beakers. 3. Repeat this process 4 more mes (for a total of 5 trials).

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Ques ons
1. What observa ons can you make regarding the gene pool and gene frequency of the founding individuals?

2. Do these results vary between the popula ons founded by beakers #1, #2 and #3? Why or why not?

3. What observa ons can you make about the gene c varia on between the parent and founding popula ons?

Experiment 4: Muta ons
Many stochas c events change both the gene pool and gene frequencies over me. These parameters can also change as a result of muta on and natural selec on. Muta ons are a change in the sequence of DNA. Most muta ons do not change the phenotype and confer no advantages or disadvantages to the individual. Each of us has hundreds and probably thousands of muta ons that do not affect our fitness. To answer the assessments for this exercise, assume the following:

• • •

There are approximately 3,000,000,000 base pairs in the mammalian genome (genes cons tute only a small por on of this total). There are approximately 10,000 genes in the mammalian genome. A single gene averages 10,000 base pairs in size.

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Ques ons
1. How many total base pairs are in all the mammalian genes?

2. What propor on (%) of the total genome does this represent?

3. What is the probability that a random muta on will occur in any given gene?

4. Only 1 out of 3 muta ons that occur in a gene result in a change to the protein structure. What is the probability that a random muta on will change the structure of a protein?

Some muta ons do change the protein coded for by a gene. The vast majority of these muta ons are lethal and the embryo never fully develops. Occasionally muta ons do not effect embryonic development and the offspring is born without complica on. Natural selec on is a selec on pressure that acts on phenotypes in one of three ways:

• • •

It will confer an adap ve advantage, an adap ve disadvantage, or remain en rely neutral. A classic example to illustrate natural selec on comes from England.

Prior to the Industrial Revolu on the na ve moths were predominantly a light color, though darker versions of the same species existed. The lighter color blended with the light bark of the local trees, while the darker moths experienced a higher preda on rate – they were easier for birds to spot and fewer survived to reproduce. As England entered the Industrial Revolu on they began burning fossil fuels with li le regard to the pollutants they were emi ng. The trunks of the trees became coated with soot and the color darkened. The lighter moths became more conspicuous and the darker were be er camouflaged. The propor on of white to dark moths changed.

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Lab 15: Popula on Gene cs
Experiment 5: Natural selec on

Materials
Red beads Blue beads 1 100mL Beaker

Note: When you are finished with this experiment, return the beads to their appropriate bags.

Procedure
1. 2. 3. 4. 5. Remove the two sheets of paper with blue and red “habitats”, at the end of this lab. Place 50 red and 50 blue beads into a 100 ml beaker. Mix them well and pour them onto the sheet marked “Red Habitat”. Keep the beads that fall onto habitat that matches their color. For each bead that you keep (and return to the beaker), add another bead of the same color to the beaker (discard the rest). 6. Repeat this three mes. 7. Record the remaining colors. Blue _____________ Red _____________ 8. With the remaining beads, repeat the process using the “Blue Habitat”. What beads remain? Blue _____________ Red _____________

Ques ons
1. Do you observe a selec ve advantage or disadvantage for the red or blue beads on the blue habitat? Why?

2. How did the distribu on of phenotypes change over me?

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3. What results would you predict if star ng with the following popula on sizes? 1000 100 10

Experiment 6: Sickle cell anemia inheritance pa erns
Facts:

• • • • •

Sickle Cell Anemia is a gene c disease (1 base pair muta on that changed a protein). It is common in those of African ancestry. S will represent the normal dominant allele and s for the recessive sickle allele. They are co-dominant alleles. SS is normal, Ss is not fatal, ss is debilita ng, painful and o en fatal.

Materials
Red beads Blue beads 1 100mL Beaker

Procedure
1. Place 25 Red (S) and 25 Blue (s) beads into the 100 ml beaker and mix well. 2. Randomly (without looking) remove 2 beads. Repeat 10 mes (without returning the beads to the beaker), each me recording if it was a SS, Ss or ss.

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3. Remove each ss from the popula on – they died. 4. The remaining beads survived and reproduced. 5. Count how many red and blue beads remained (separately) and place twice that number back in the beaker. 6. Repeat the process 7 mes.

Ques ons
1. What is the remaining ra o of alleles?

2. Have any been selected against?

3. Given enough genera ons, would you expect one of these alleles to completely disappear from the popula on? Why or why not?

4. Would this be different if you started with a larger popula on? Smaller?

5. A er hundreds or even thousands of genera ons both alleles are s ll common in those of African Ancestry. How would you explain this?

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6. The worldwide distribu on of sickle gene matches very closely to the worldwide distribuon of Malaria.

Sickle gene distribu on; image courtesy of en.wikipedia under the GNU Free Documenta on License.

Malaria distribu on; image courtesy of en.wikipedia under the GNU Free Documenta on License. Is this significant? Why or why not?

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Introductory Biology

Appendix Good Laboratory Techniques
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Appendix: Good Lab Techniques Good Laboratory Techniques
Science labs, whether at universi es or in your home, are places of adventure and discovery. One of the first things scien sts learn is how exci ng experiments can be. However, they must also realize science can be dangerous without some instruc on on good laboratory prac ces.

• Read the protocol thoroughly before star ng any new experiment. You should be familiar with the ac on required every step of the way.

• Keep all work spaces free from clu er and dirty dishes. • Read the labels on all chemicals, and note the chemical safety ra ng on each container. Read all MSDS (provided on www.eScienceLabs.com).

• Thoroughly rinse labware (test tubes, beakers, etc.) between experiments. To do so, wash with a soap and hot water solu on using a bo le brush to scrub. Rinse completely at least four mes. Let air dry

• Use a new pipet for each chemical dispensed. • Wipe up any chemical spills immediately. Check MSDSs for special handling instruc ons (provided on www.eScienceLabs.com).

A benchcoat will prevent any spilled liquids from contamina ng the surface you work on.

A

B

C

Special measuring tools in make experimenta on easier and more accurate in the lab. A shows a beaker, B graduated cylinders, and C test tubes in a test tube rack.

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Appendix: Good Lab Techniques
• •
Use test tube caps or stoppers to cover test tubes when shaking or mixing – not your finger! When preparing a solu on, refer to a protocol for any specific instruc ons on prepara on. Weigh out the desired amount of chemicals, and transfer to a beaker or graduated cylinder. Add LESS than the required amount of water. Swirl or s r to dissolve the chemical (you can also pour the solu on back and forth between two test tubes), and once dissolved, transfer to a graduated cylinder and add the required amount of liquid to achieve the final volume.

• A molar solu on is one in which one liter • Ex:

Disposable pipets aid in accurate measuring of small volumes of liquids. It is important to use a new pipet for each chemical to avoid contamina on.

(1L) of solu on contains the number of grams equal to its molecular weight.

1M = 11g CaCl x 110g CaCl/mol CaCl (The formula weight of CaCl is 110g/mol)

• A percent solu on can be prepared by percentage of weight of chemical to 100ml of solvent (w/v) , or volume of chemical in 100ml of solvent (v/v).

• Ex:
20g NaCl + 80ml H2O = 20% w/v NaCl solu on

• Concentrated solu ons, such as 10X, or ten mes the normal strength, are diluted such that the final concentra on of the solu on is 1X.

• Ex:
To make a 100ml solu on of 1X TBE from a 10X solu on: 10ml 10X TBE + 90ml water = 100ml 1X TBE

• Always read the MSDS before disposing of a chemical to insure it does not require extra measures. (provided on www.eScienceLabs.com)

• Don’t pour unused chemical back into the original bo le. • Avoid prolonged exposure of chemicals to direct sunlight and extreme temperatures. 182

Appendix: Good Lab Techniques Lab 22: Plant Reproduc on
• Immediately secure the lid of a chemical a er use. • Prepare a dilu on using the following equa on:

Where c1 is the concentra on of the original solu on, v1 is the volume of the original soluon, and c2 and v2 are the corresponding concentra on and volume of the final solu on. Since you know c1, c2, and v2, you solve or v1 to figure out how much of the original soluon is needed to make a certain volume of a diluted concentra on.

•

If you are ever required to smell a chemical, always wa a gas toward you, as shown in the figure below.. This means to wave your hand over the chemical towards you. Never directly smell a chemical. Never smell a gas that is toxic or otherwise dangerous.

• • • • •

Use only the chemicals needed for the ac vity. Keep lids closed when a chemical is not being used. When dilu ng an acid, always pour the acid into the water. Never pour water into an acid. Never return excess chemical back to the original bo le. This can contaminate the chemical supply. Be careful not to interchange lids between different chemical bo les.

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Appendix: Good Lab Techniques Lab 28: Ecological Interac ons
• • •
When pouring a chemical, always hold the lid of the chemical bo le between your fingers. Never lay the lid down on a surface. This can contaminate the chemical supply. When using knives or blades, always cut away from yourself. Wash your hands a er each experiment.

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1500 West Hampden Avenue Building 5 Unit H Sheridan, CO 80110 888.375.5487 • www.esciencelabs.com
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